Artificial tissue construct and method for producing the same

ABSTRACT

An object of the present invention is to provide an artificial tissue construct that has means for transporting nutrients, oxygen, waste products, or the like and is viable in vivo. The present invention relates to a tissue construct formed in vitro, which comprises a vascular layer, a basal membrane layer, and a tissue-forming cell layer.

This is a divisional of U.S. patent application Ser. No. 11/628,054filed Nov. 30, 2006, which is a 371 National Stage Entry ofPCT/JP2005/010307 filed May 31, 2005, which claims priority fromJapanese Patent Application No. 2004-163512 filed Jun. 1, 2004, thecontents of all of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a tissue construct formed in vitro,which comprises a vascular layer, a basal membrane layer, and atissue-forming cell layer, a laminated tissue construct comprising thetissue constructs, and a method for producing them.

BACKGROUND ART

Recently, technology for direct transplantation of artificial alternatesor cell tissues obtained by culturing cells has been a focus ofattention. Typical examples of such technology include artificial skin,artificial blood vessels, and cultured cell tissues. Artificial skin orthe like containing a synthetic polymer is not preferable fortransplantation because it may cause rejection or other problems. On theother hand, with a cultured cell tissue, there is no concern aboutrejection, because such tissue is obtained by culturing the cells of asubject into which the tissue will be transplanted and thus it ispreferable for transplantation. Such cultured cell tissue is prepared bycollecting cells from a subject for transplantation and then culturingthe cells.

Many animal cells have adhesion dependency (cells grow while adhering tosomething). Animal cells are thus unable to survive for a long timeperiod in a floating state ex vivo. In a cell culture for preparing theabove cell tissue, for example, a polymer material such as modifiedpolystyrene having enhanced cell adhesiveness through surface treatmentor a culture plate that is prepared by uniformly applying a celladhesive protein (e.g., collagen) or a cell-adhesive polymer (e.g.,poly-L-lysine) to glass or a polymer material has been used as acarrier. Cells that have adhered to and grown on such carrier in aplanar state grow under a culture environment while formingextracellular matrices comprising proteins and carbohydrates. Ingeneral, it is required to harvest such cultured cells through treatmentwith proteinase such as trypsin or a chemical drug. Thus, such processis problematic in that treatment steps become complicated, thepossibility of contamination becomes high, cells are denatured ordamaged, original cellular functions may deteriorate, and the like.

Accordingly, JP Patent Publication (Kokai) No. 2003-38170 A discloses amethod for producing a cell sheet, which comprises preparing a cellculture support obtained by patterning a temperature-responsive polymeron a culture base, culturing cells on the cell culture support, causingthe support to closely adhere to a polymer film by varying temperature,and removing the cells together with the polymer film from the supportwithout damaging the cells, so as to produce a cell sheet.

However, transplantation or the like of such cell sheet obtained by themethod disclosed herein into a living body is problematic in that anartificial tissue having a thickness of approximately several hundredmicrons prepared through multiple lamination of such multiple cellsheets lacks means for transporting nutrients, oxygen, and wasteproducts, and thus it causes cell necrosis within the artificial tissueconstruct.

It is possible to form an artificial tissue construct with a thicknessthat does not cause cell necrosis within the tissue, followed bytransplantation thereof into a living body, so as to cause angiogenesisto take place in vivo. However, such means is unrealistic, because fewcells can be transplanted at a time by such means, and suchtransplantation should be carried out repeatedly to restore organfunctions where there has been damage on a large scale.

Furthermore, a method for constructing an artificial organ using aspecific cell culture method is also known (JP Patent Publication(Kokai) 2003-24351 A). With this method, an artificial blood vessel isformed by adhesion of vascular endothelial cells or the like to atubular cell culture substrate. However, in order to prepare manyartificial blood vessels by this method, many finely processed cellculture substrates must be prepared and tissue formation requires muchtime. Thus, such method has also low industrial productivity.

DISCLOSURE OF THE INVENTION

The present invention has been completed to address the above problemsin the above conventional technology. Specifically, an object of thepresent invention is to provide an artificial tissue construct that hasmeans for transporting nutrients, oxygen, waste products, or the likeand is viable in vivo.

As a result of intensive studies to achieve the above object, thepresent inventors have discovered that an artificial tissue constructhaving blood vessel tissues can be produced by laminating together atleast one layer of each of vascular, basal membrane, and tissue-formingcell layers. Thus, the present inventors have completed the presentinvention.

The present invention encompasses the following invention.

(1) A tissue construct formed in vitro, which comprises a vascularlayer, a basal membrane layer, and a tissue-forming cell layer.

(2) The tissue construct according to (1), wherein the basal membranelayer is present on the tissue-forming cell layer and the vascular layeris present on the basal membrane layer.

(3) A laminated tissue construct formed in vitro, wherein vascular,basal membrane, and tissue-forming cell layers are laminated togetherand which comprises at least one layer of each of these 3 types oflayers.

(4) The laminated tissue construct according to (3), wherein a basalmembrane layer is present on a tissue-forming cell layer, a vascularlayer is present on a basal membrane layer, and a tissue-forming celllayer is present on a basal membrane layer or a vascular layer.(5) A method for producing a tissue construct comprising atissue-forming cell layer, a basal membrane layer, and a vascular layer,which comprises the steps of:(a) forming a tissue-forming cell layer on a culture base;(b) forming a basal membrane layer on the obtained tissue-forming celllayer;(c) causing angiogenic cells to adhere to regions having good celladhesiveness on the surface of a cell array substrate having a celladhesiveness variation pattern that comprises regions having good celladhesiveness and regions having inhibited cell adhesiveness,transferring the adhered cells onto the basal membrane layer in suchpatterned state, and culturing the transferred cells; and(d) separating and collecting a tissue construct comprising thetissue-forming cell layer, the basal membrane layer, and the vascularlayer from the culture base.(6) A method for producing a laminated tissue construct, which compriseslaminating together tissue constructs produced by the method accordingto (5).(7) A method for producing a laminated tissue construct, which comprisesproducing a first tissue construct by a method comprising the followingsteps (a) to (d):(a) forming a tissue-forming cell layer on a culture base;(b) forming a basal membrane layer on the obtained tissue-forming celllayer;(c) causing angiogenic cells to adhere to regions having good celladhesiveness on the surface of a cell array substrate having a celladhesiveness variation pattern that comprises regions having good celladhesiveness and regions having inhibited cell adhesiveness,transferring the adhered cells onto the basal membrane layer in suchpatterned state, and culturing the transferred cells; and(d) separating and collecting a first tissue construct comprising thetissue-forming cell layer, the basal membrane layer, and the vascularlayer from the culture base, producing a second tissue construct by amethod comprising the following steps (e) to (f):(e) forming a tissue-forming cell layer on a culture base;(f) forming a basal membrane layer on the obtained tissue-forming celllayer; and(g) separating and collecting the tissue-forming cell layer and thebasal membrane layer from the culture base, andlaminating together the 1^(st) and the 2^(nd) tissue constructs.(8) The method according to (6) or (7), which further comprises the stepof transporting a culture solution to the vascular layer within thelaminated tissue construct.(9) The method according to any one of (5) to (8), wherein the celladhesiveness variation layer is a photocatalyst-comprising celladhesiveness variation layer that comprises a photocatalyst and the celladhesiveness variation material.(10) The method according to any one of (5) to (8), wherein the celladhesiveness variation layer comprises a photocatalyst treatment layerthat comprises a photocatalyst and a cell adhesiveness variationmaterial layer that comprises the cell adhesiveness variation materialformed on the photocatalyst treatment layer.(11) The method according to (9), by which the cell adhesivenessvariation pattern is formed by arranging the cell adhesiveness variationlayer that comprises the cell adhesiveness variation material and thephotocatalyst-comprising layer so that the layers face each other, andthen carrying out energy irradiation.(12) The method according to any one of (5) to (11), wherein the celladhesiveness variation pattern is a pattern wherein linear regionshaving good cell adhesiveness and spaces comprised of the regions havinginhibited cell adhesiveness are arranged alternately, the line widths ofthe regions having good cell adhesiveness are each between 20 μm and 200μm, and the space widths between such lines are each between 100 μm and1000 μm.(13) The method according to any one of (5) to (12), wherein the culturebase has a surface that is capable of retaining cells with weakadhesiveness.(14) A method for regenerating a tissue, which comprises transplantingthe tissue construct according to any one of (1) to (4).(15) A tissue construct comprising a vascular layer, a basal membranelayer, and a tissue-forming cell layer, wherein the basal membrane layeris formed covering almost the entire surface of the vascular layerformation region of the tissue-forming cell layer.(16) A laminated tissue construct, which comprises vascular, basalmembrane, and tissue-forming cell layers and comprises at least onelayer of each of these 3 types of layers, wherein a basal membrane layeris formed covering almost the entire surface of the vascular layerformation region of a tissue-forming cell layer.(17) The laminated tissue construct according to (16), wherein a basalmembrane layer is present on a tissue-forming cell layer, a vascularlayer is present on a basal membrane layer, and a tissue-forming celllayer is present on a basal membrane layer or a vascular layer.

According to the present invention, an artificial tissue construct isprovided, which has means for transporting nutrients, oxygen, wasteproducts, or the like and is viable in vivo.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2004-163512, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an example of a step in the method for producing the cellarray substrate of the present invention.

FIG. 2 shows another example of a step in the method for producing thecell array substrate of the present invention.

FIG. 3 shows another example of a step in the method for producing thecell array substrate of the present invention.

FIG. 4 is a schematic sectional view showing an example of thephotocatalyst-comprising-layer-side base plate used in the presentinvention.

FIG. 5 is a schematic sectional view showing another example of thephotocatalyst-comprising-layer-side base plate used in the presentinvention.

FIG. 6 is a schematic sectional view showing another example of thephotocatalyst-comprising-layer-side base plate used in the presentinvention.

FIG. 7 shows an embodiment of a step for causing angiogenic cells toadhere to a cell array substrate in a pattern and transferring theadhered angiogenic cells to the basal membrane layer.

FIG. 8 is a schematic view showing an example of the method of thepresent invention.

FIG. 9 is a photograph showing cells arrayed on a cell array substrate.

FIG. 10 shows an embodiment of a method for producing in vitro a tissueconstruct comprising a vascular layer, a basal membrane layer, and atissue-forming cell layer.

FIG. 11 shows an embodiment of a method for producing in vitro alaminated tissue construct in which vascular layers, basal membranelayers, and tissue-forming cell layers are laminated together.

EXPLANATION OF SYMBOLS

1: substrate, 2: photocatalyst-containing cell adhesiveness variationlayer, 3: substrate for pattern formation, 4: photomask, 5: energy, 6:cell adhesiveness variation pattern, 15: cell array substrate, 16: basalmembrane layer, 17: region having good cell adhesiveness, 18: regionhaving inhibited cell adhesiveness, 19: cell, 20: water-repellentmaterial, 21: cell-adhesive material, 22: cell stimulating factor, 101:tissue-forming cell, 102: culture base, 103: tissue-forming cell layer,104: basal membrane layer, 105: cell array substrate, 106: angiogeniccells, 107: tissue construct, 108: vascular layer, 109: laminated tissueconstruct in which 1^(st) tissue constructs are laminated together, and110: laminated tissue construct in which 1^(st) and 2^(nd) tissueconstructs are laminated together

The present invention will be described in detail below.

The present invention relates to a tissue construct formed in vitro,which comprises a vascular layer, a basal membrane layer, and atissue-forming cell layer. “Vascular layer” means a layer containingangiogenic cells. Examples of angiogenic cells include vascularendothelial cells, smooth muscle cells, and mural cells. Vascularendothelial cells are preferable to avoid coagulation of bloodcomponents in blood vessels for smooth blood flow. Furthermore,angiogenic cells are desirably composed of vascular endothelial cellstogether with smooth muscle cells or mural cells in order to maintainfunctions and structures of the vascular endothelial cells. In thevascular layer, it is preferable that angiogenic cells be arrayed in apattern. A pattern to be formed is not particularly limited, as long asit is a two-dimensional pattern. For example, a linear, a mesh, acircular, or a quadrille pattern, a pattern wherein the inside of eachfigure (e.g., circle or tetragram) is filled with cells, or the like canbe formed. A linear or a mesh pattern is preferable. When angiogeniccells are arrayed in a linear or a mesh pattern and then cultured,tissue formation is promoted and angiogenesis is thus promoted. Hence,the vascular layer in the present invention preferably includes bloodvessel tissue.

In the present invention, “basal membrane layer” means a layer thatcomprises a basal membrane constitutive protein as a major component. A“basal membrane layer” is an extracellular matrix in the form of alayer, which unites cell growth factors that stimulate cellularactivity, a vascular layer, and a tissue-forming cell layer, so as toform an aggregate, and it contains collagen, fibronectin, and laminin.The basal membrane layer may be an extract from a living body or may beproduced by cells. Furthermore, the basal membrane layer may also beformed with the addition of artificial substances.

“Tissue-forming cell layer” means a layer containing tissue-formingcells. “Tissue-forming cells” means cells having functions required fora tissue construct that is constituted in vitro. Examples oftissue-forming cells include organ cells. Specific examples of suchorgan cells include cells derived from organs of the metabolic system,such as hepatic parenchymal cells and pancreatic β cells, and cellsderived from organs of the structural system, such as epithelial cellsof the skin. In the above tissue construct, preferably, the basalmembrane layer is present on the tissue-forming cell layer and thevascular layer is present on the basal membrane layer.

The present invention also relates to a laminated tissue construct,which is formed in vitro by laminating together vascular, basalmembrane, and tissue-forming cell layers and comprises at least onelayer of each of these 3 types of layers. In the laminated tissueconstruct, preferably, a basal membrane layer is present on atissue-forming cell layer, a vascular layer is present on a basalmembrane layer, and a tissue-forming cell layer is present on a basalmembrane layer or a vascular layer. Specifically, the laminated tissueconstruct has a structure in which a three-layer structure and atwo-layer structure are laminated together. In the three-layerstructure, a basal membrane layer is present on a tissue-forming celllayer and a vascular layer is present on a basal membrane layer. In thetwo-layer structure, a basal membrane layer is present on atissue-forming cell layer.

At least 1 and preferably 1 to 5 pieces of three-layer structures eachhaving a vascular layer may be present in the laminated tissueconstruct. In the case of the two-layer structure wherein the basalmembrane layer is present on the tissue-forming cell layer, generally 0to 5 two-layer structures are present. Generally 5 to 10 structures arepresent in total, including two-layer and three-layer structures.

The above basal membrane layer is formed covering almost the entiresurface of a region on the above tissue-forming cell layer on which avascular layer is formed. “Almost the entire surface” means generally90% or more and preferably 95% of the surface.

The present invention also relates to a method for producing in vitro atissue construct comprising a vascular layer, a basal membrane layer,and a tissue-forming cell layer. The tissue construct can be produced bya method comprising the following steps (a) to (d):

(a) forming a tissue-forming cell layer on a culture base;

(b) forming a basal membrane layer on the obtained tissue-forming celllayer;

(c) causing angiogenic cells to adhere to regions having good celladhesiveness on the surface of a cell array substrate having a celladhesiveness variation pattern that comprises regions having good celladhesiveness and regions having inhibited cell adhesiveness,transferring the adhered cells onto the basal membrane layer in suchpatterned state, and culturing the transferred cells; and(d) separating and collecting a first tissue construct comprising thetissue-forming cell layer, the basal membrane layer, and the vascularlayer from the culture base.

FIG. 10 shows an embodiment of the above production method. In FIG. 10,101 denotes tissue-forming cells, 102 denotes a culture base, 103denotes a tissue-forming cell layer, 104 denotes a basal membrane layer,105 denotes a cell array substrate, 106 denotes angiogenic cells, 107denotes a formed tissue construct, and 108 denotes a vascular layer.

The culture base is not particularly limited, as long as atissue-forming cell layer can be formed thereon. Preferably, a culturebase from which a tissue construct can be separated without damagingsuch cell layer is used. Examples of such culture base include culturebases having surfaces capable of retaining cells with weak adhesiveness,such as a culture base prepared by subjecting a polystyrene substrate toweak plasma treatment for cell adhesion and a culture base prepared byintroducing a small amount of a material such as 2-methacryloyloxyethylphosphorylcholine or fluoroalkylsilane having property of inhibitingcell adhesion onto a substrate surface. Examples of such method forintroducing a small amount of such material include a method thatinvolves sufficiently introducing a material to a substrate throughadsorption treatment or the like and then carrying out decompositionthrough UV treatment, ozone treatment, or plasma treatment and a methodthat involves coating with a solution in which a material is slightlydissolved to form a thin layer. The proportions of materials to beintroduced differ and should be adjusted depending on cell types to becaused to adhere and material types to be introduced onto substrates.

Furthermore, there exist temperature-responsive polymer materials, suchas poly-N-isopropylacrylamide. This material possesses hydrophobicity;that is, cell adhesiveness, under environment where the temperature isat a phase transition temperature or higher, but becomes hydrophilic ata phase transition temperature or lower so as to lose its celladhesiveness. A cell base prepared by polymerizing such material on apolymer or a glass substrate is also an example. In the presentinvention, poly-N-isopropylacrylamide is preferably used.

The tissue-forming cell layer can be formed by a cell culture methodthat is generally employed in the technical field. For example, atissue-forming cell layer can be formed by inoculating cells on aculture base at a density between 10⁴ and 10⁸ cells/cm² and thenculturing the cells at 37° C. for 30 minutes to 48 hours. As a medium, amedium that is generally used in the technical field can be used.Examples of such a medium that can be used herein include an MEM medium,a BME medium, a DME medium, an αMEM medium, an IMEM medium, an ESmedium, a DM-160 medium, a Fisher medium, an F12 medium, a WE medium, anRPMI medium, any one of these media supplemented with a serum component(e.g., fetal calf serum), and a commercial serum-free medium.

The basal membrane layer can also be formed by culturing tissue-formingcells, or it may also be formed by adding a matrix. A matrix to be addedherein is not particularly limited, as long as it contains collagen,laminin, or fibronectin. An example of such matrix is prepared by addingmaterials derived from a living body, such as collagen and laminin, toan artificial polymer material such as GFR Matrigel (e.g., Mebiol gel).In a case where a matrix is added, a basal membrane layer can be formedby carrying out incubation at approximately 37° C. for several hoursafter addition.

The vascular layer can be formed by causing the surface of a cell arraysubstrate on which angiogenic cells are arrayed in a pattern to comeinto contact with the basal membrane layer, culturing the cells to formtissues, and then removing the cell array substrate. Culture conditionsthat are generally employed in the technical field can be employed. Forexample, culture may be carried out at 37° C. for generally 2 to 48hours and preferably for 4 to 24 hours.

A cell array substrate having a cell adhesiveness variation pattern thatcomprises regions having good cell adhesiveness and regions havinginhibited cell adhesiveness and adhesion of angiogenic cells to theregions having good cell adhesiveness on the surface are describedlater. Finally, the tissue construct comprising the tissue-forming celllayer, the basal membrane layer, and the vascular layer is separated andcollected from the culture base.

The present invention further relates to a method for producing in vitroa laminated tissue construct in which vascular, basal membrane, andtissue-forming cell layers are laminated together. The thus producedlaminated tissue construct comprises at least one layer of each of these3 types of layers. The laminated tissue construct can be produced byproducing 1^(st) tissue constructs each comprising a vascular layer, abasal membrane layer, and a tissue-forming cell layer, and thenlaminating together the thus produced 1^(st) tissue constructs.Alternatively, the laminated tissue construct can also be produced byproducing the above 1^(st) tissue construct, producing a 2^(nd) tissueconstruct comprising a basal membrane layer and a tissue-forming celllayer by a method comprising the following steps (e) to (g):

(e) forming a tissue-forming cell layer on a culture base;

(f) forming a basal membrane layer on the obtained tissue-forming celllayer; and

(g) separating and collecting the tissue-forming cell layer and thebasal membrane layer from the culture base; and then

laminating together the 1^(st) and 2^(nd) tissue constructs.

FIG. 11 shows an embodiment of the above production method. In FIG. 11,107 denotes a tissue construct, 108 denotes vascular layers, 109 denotesa laminated tissue construct produced by laminating 1^(st) tissueconstructs, and 110 denotes a laminated tissue construct produced bylaminating together the 1^(st) and 2^(nd) tissue constructs.

In the production of the 2^(nd) tissue construct, a culture base, atissue-forming cell layer, and the basal membrane layer are formed in amanner similar to that in the production of the above 1^(st) tissueconstruct.

Here, the order of laminating together the 1^(st) and 2^(nd) tissueconstructs is not particularly limited. 1 to 6 and preferably 2 to 3pieces of 2^(nd) tissue constructs having no vascular layers arepreferably present per 1 layer of the 1^(st) tissue construct.

After laminating such tissue constructs, preferably, a culture solutionis transported to the vascular layers within the laminated tissueconstruct. This promotes the tissue formation by angiogenic cellsarrayed in a pattern on each of the vascular layers.

I. Cell Array Substrate

The cell array substrate in the present invention is characterized inhaving a cell adhesiveness variation pattern that comprises regionshaving good cell adhesiveness and regions having inhibited celladhesiveness patterned on a substrate.

“Cell adhesiveness” means strength for the adhesion of cells; that is,the degree of ease with which cells adhere. “Regions having good celladhesiveness” means regions wherein cell adhesiveness is good. “Regionshaving inhibited cell adhesiveness” means regions wherein celladhesiveness is poor. Accordingly, when cells are inoculated on suchcell array substrate having a cell adhesiveness variation pattern, cellsadhere to the regions having good cell adhesiveness, but no cells adhereto the regions having inhibited cell adhesiveness. Hence, cells arearrayed in a pattern on the surface of the cell array substrate.

Cell adhesiveness can differ depending on cells that are caused toadhere. Hence, “good cell adhesiveness” means that cell adhesiveness fora specific type of cell is good. Therefore, a plurality of regionshaving good cell adhesiveness are present corresponding to a pluralityof types of cells on a cell array substrate. Specifically, regionshaving good cell adhesiveness, which vary in cell adhesiveness (2 ormore different levels) may be present on the cell array substrate.

The cell adhesiveness variation pattern is, for example, formed byforming a cell adhesiveness variation layer that comprises a celladhesiveness variation material whose cell adhesiveness is varied alongwith energy irradiation on a substrate, varying cell adhesivenessthrough energy irradiation on specific regions, and then forming apattern wherein regions differ in cell adhesiveness. Examples of suchmaterial whose cell adhesiveness is varied include both a material whosecell adhesiveness is acquired or increased along with energy irradiationand a material whose cell adhesiveness is decreased or disappears alongwith energy irradiation.

A substrate used for the cell array substrate of the present inventionis not particularly limited, as long as it is formed of a materialcapable of forming a cell adhesiveness variation pattern on its surface.Specific examples of such substrate include inorganic materials such asmetal, glass, and silicon, and organic materials represented by plastic.The shape of such material is also not limited. Examples of such shapeinclude a flat plate, a flat membrane, a film, and a porous membrane.

The cell adhesiveness variation material and the cell adhesivenessvariation layer will be explained in an embodiment using aphotocatalyst.

Another example included herein is a cell adhesiveness variation patternthat is formed of a cell-adhesion inhibiting layer that comprises acell-adhesion-inhibiting material having low cell adhesiveness and acell adhesion layer that is formed on the cell-adhesion-inhibiting layerand comprises a cell adhesive material having cell adhesiveness. Here,the cell adhesion layer is decomposed and then disappears along withenergy irradiation to cause the cell-adhesion-inhibiting layer to beexposed, so that regions differing in cell adhesiveness are formed.Similarly, another example included herein is a cell adhesivenessvariation pattern that is formed of a cell adhesion layer and acell-adhesion-inhibiting layer formed on the cell adhesion layer,wherein the cell-adhesion-inhibiting layer is decomposed and thendisappears along with energy irradiation to cause the cell adhesionlayer to be exposed, so that regions differing in cell adhesiveness areformed.

Examples of such cell adhesive material include extracellular matricessuch as various types of collagen, fibronectin, laminin, vitronectin,and cadherin, and RGD peptide. Another example of the same is apolyolefin resin wherein a carbonyl group or a carboxyl group has beenintroduced by a technique such as plasma treatment, corona treatment,ion beam irradiation treatment, or electron beam irradiation treatmentin order to impart cell adhesiveness. Examples of suchcell-adhesion-inhibiting material include fluorine materials such aspolytetrafluoroethylene (PTFE), polyimide, and phospholipid. Moreover,through the use of a method such as an inkjet method, a cell adhesivematerial may be put on a cell-adhesion-inhibiting layer to form apattern or a cell-adhesion-inhibiting material may be put on a celladhesion layer to form a pattern. Alternatively, a cell adhesivenessvariation pattern that comprises regions where a cell adhesive materialis present (regions having good cell adhesiveness) and regions where acell adhesive material is absent (regions having inhibited celladhesiveness) can also be formed by: forming a layer that comprises anaffinity variation material whose affinity for a cell adhesive materialis varied along with energy irradiation on a substrate; forming apattern through energy irradiation that comprises regions havingaffinity for the cell adhesive material and regions lacking suchaffinity; introducing a solution that contains the cell adhesivematerial; and then washing. In such embodiment, a pattern can be formedusing a cell adhesive material that cannot be directly patterned on asubstrate. For example, as shown in FIG. 8, a pattern that comprisesregions (20) comprising a layer that comprises a water repellentmaterial and regions lacking such material is formed on a hydrophilicsubstrate (1) such as glass. A hydrophilic cell adhesive material (21)that is hardly adsorbed to the water repellent material is introducedthereto. The substrate is then washed. Accordingly, regions wherein thehydrophilic cell adhesive material is present (regions having good celladhesiveness) and regions wherein the water repellent material ispresent (regions having inhibited cell adhesiveness) form a pattern. Anextracellular matrix such as collagen can be used as such hydrophiliccell adhesive material to be used in this case.

In the present invention, cells arrayed in a pattern on a cell arraysubstrate are transferred to a basal membrane layer. Hence, the celladhesiveness of the above regions having good cell adhesiveness ispreferably at a proper strength. With such proper adhesion strength, itbecomes possible to easily transfer the cells to a basal membrane layer,while forming a cell pattern by adhering cells only to specific regions.Therefore, it is preferable that the cell adhesiveness of regions havinggood cell adhesiveness on a cell array substrate is higher than those ofregions having inhibited cell adhesiveness, but lower than that of thebasal membrane layer.

Such cell adhesiveness can be evaluated using a water contact angle onthe surface. It is preferable that regions having good cell adhesivenessof the cell adhesiveness variation pattern in the present invention eachhave a water contact angle between 10° and 40°. When cells are caused toadhere to a cell array substrate and then transferred to a basalmembrane layer with a water contact angle within such range, the cellscan be caused to adhere to a cell array substrate in the form of amonolayer. And then, the cells can be easily transferred to a basalmembrane layer because of weak adhesiveness to the cell array substrate.“Contact angle” means an angle formed by the surface of a liquid and thesurface of a solid where the free surface of the liquid at rest comesinto contact with the wall of the solid (angle measured within theliquid).

The term “water contact angle” used herein means a value measured usinga static contact angle measurement method. The above water contact angleis generally observed by adding minute waterdrops dropwise to thesurface of a material under atmospheric pressure using an instrumentsuch as a syringe and then observing the angle formed by theliquid/vapor interface (at the droplet end) and the surface of a solidusing a magnifying glass or the like.

There is no particular limitation of a means for forming the above celladhesiveness variation pattern that comprises regions having good celladhesiveness and regions having inhibited cell adhesiveness arranged ina pattern. Examples of such means include various printing methods suchas a gravire printing method, a screen printing method, an offsetprinting method, a flexographic printing method, and a contact printingmethod, a method using various lithographic methods, a method based onan inkjet method, and three-dimensional modeling such as carving finegrooves. In the present invention, a lithographic method using aphotocatalyst is preferable. Specifically, in such method, a celladhesiveness variation material (whose cell adhesiveness is varied bythe action of a photocatalyst along with energy irradiation) and thephotocatalyst are used and energy irradiation that is carried outaccording to a required pattern, so as to form a required celladhesiveness variation pattern. In such embodiment, a high-definitionpattern can be formed with convenient steps without using any treatmentsolution that adversely affects cells. Moreover, such embodiment doesnot require any modification of a cell adhesiveness variation material.Thus, options for material selection can be expanded. Furthermore, abiological cell adhesiveness variation material that exerts specificadhesiveness described later can also be used without any problems.

A pattern to be formed is not particularly limited, as long as it is atwo-dimensional pattern. Such pattern is designed according to thepattern of angiogenic cells, which is formed within a vascular layer ina tissue construct. Hence, it is preferable to form a pattern thatenables cells to adhere in a linear or mesh pattern. When such linear ormesh pattern is formed, the line width in the pattern is generallybetween 20 μm and 200 μm and preferably between 50 μm and 100 μm. Inparticular, when capillary vessels are formed by arranging and culturingvascular endothelial cells in a line-shaped pattern, it is preferablethat a cell adhesiveness variation pattern is formed where linearregions having good cell adhesiveness and spaces comprised of regionshaving inhibited cell adhesiveness are arranged alternately, so as tocause the vascular endothelial cells to adhere to form a linear pattern.In such embodiment, a pattern is preferably formed wherein cells arecaused to adhere so that a line width can contain 1 to 10 cells andpreferably 1 to 5 cells. Specifically, the line width of a region havinggood cell adhesiveness is generally between 20 μm and 200 μm, andpreferably between 50 μm and 80 μm. The space widths between such lines(the spaces comprised of regions having inhibited cell adhesiveness) areeach generally between 100 μm and 1000 μm and preferably between 400 μmand 800 μm. With a line width determined within the above numericalrange, vascular endothelial cells can efficiently form a tubular tissue.

Through the formation of such cell adhesiveness variation pattern,vascular endothelial cells caused to adhere and then transferred in alinear pattern efficiently form a tissue; that is, a linear capillaryvessel. When a cell pattern where a plurality of lines are arrangedwithout crossing each other is formed, the space widths between thelines on which cells adhere are each set to be equal to or above aspecific value as described above. Accordingly, the cells can beprevented from extending pseudopodia between the lines at the time oftheir tissue formation, which would distort the lines.

Examples of the above cell array substrate prepared by a lithographicmethod using a photocatalyst include the following three embodiments.Each of these embodiments will be described as follows.

A. 1^(st) Embodiment

A 1^(st) embodiment of the cell array substrate of the present inventionis a cell array substrate comprising a cell adhesiveness variation layerthat is formed on a substrate and comprises a cell adhesivenessvariation material whose cell adhesiveness is varied by the action of aphotocatalyst along with energy irradiation, wherein the above celladhesiveness variation layer forms a cell adhesiveness variation patternwith variations in cell adhesiveness characterized in that the abovecell adhesiveness variation layer is a photocatalyst-comprising celladhesiveness variation layer that comprises the photocatalyst and theabove cell adhesiveness variation material.

In this embodiment, the cell adhesiveness variation layer is aphotocatalyst-comprising cell adhesiveness variation layer thatcomprises the photocatalyst and the above cell adhesiveness variationmaterial. Thus, when energy irradiation is carried out, the celladhesiveness of the cell adhesiveness variation material is varied bythe action of the photocatalyst within the photocatalyst-comprising celladhesiveness variation layer. Hence, a cell adhesiveness variationpattern that comprises portions subjected to energy irradiation andportions not subjected to energy irradiation, where the portions differin terms of cell adhesiveness, can be formed.

Members used in such cell array substrate in this embodiment will beeach described as follows.

1. Photocatalyst-Comprising Cell Adhesiveness Variation Layer

This embodiment is characterized in that a photocatalyst-comprising celladhesiveness variation layer is formed on a substrate. Thephotocatalyst-comprising cell adhesiveness variation layer comprises atleast a photocatalyst and a cell adhesiveness variation material.

(1) Cell Adhesiveness Variation Material

A cell adhesiveness variation material used in this embodiment is notparticularly limited, as long as it is a material whose celladhesiveness is varied by the action of a photocatalyst along withenergy irradiation. Examples of such material whose cell adhesiveness isvaried include both a material that acquires or increases its celladhesiveness by the action of a photocatalyst along with energyirradiation and a material whose cell adhesiveness decreases ordisappears due to the action of a photocatalyst along with energyirradiation.

There are two major embodiments of such cell adhesiveness variationmaterial, which differ in an aspect to control cell adhesiveness. Oneembodiment is a physicochemical cell adhesiveness variation materialthat adheres to cells due to its physicochemical property and the otherembodiment is a biological cell adhesiveness variation material thatadheres to cells due to its biological property.

a. Physicochemical Cell Adhesiveness Variation Material

Examples of a physicochemical factor for causing cells to adhere to thesurface include a factor relating to surface free energy, a factorrelating to hydrophobic interaction, and the like.

A preferable physicochemical cell adhesive material havingphysicochemical cell adhesiveness due to the presence of such factorpossesses binding energy that is sufficiently high so that the mainbackbone is not decomposed by the action of a photocatalyst and also hasan organic substituent that is decomposed by the action of aphotocatalyst. Examples of such material include (1) organopolysiloxanethat is obtained through hydrolysis and polycondensation of such aschloro- or alkoxysilane using a sol-gel reaction or the like so as toexert high strength and (2) organopolysiloxane that is obtained throughcrosslinking of reactive silicones.

In the case of (1) above, a preferable organopolysiloxane is 1 or 2 ormore types of hydrolysis condensate or cohydrolysis condensate of asilicon compound that is represented by general formula:Y_(n)SiX_((4-n))(where Y indicates an alkyl group, a fluoroalkyl group, a vinyl group,an amino group, a phenyl group, or an epoxy group; X indicates analkoxyl group, an acetyl group, or halogen; and “n” is an integerbetween 0 and 3). In addition, the carbon number of a group indicatedwith Y is preferably within the range between 1 and 20. Furthermore, analkoxy group indicated with X is preferably a methoxy group, an ethoxygroup, a propoxy group, or a butoxy group.

Furthermore, polysiloxane comprising a fluoroalkyl group as an organicgroup can be particularly preferably used. Specific examples of suchpolysiloxane include hydrolysis condensate and cohydrolysis condensateof 1 or 2 or more types of the following fluoroalkyl silane. Suchpolysiloxane generally known as a fluorine silane coupling agent can beused. Examples are:

CF₃(CF₂)₃CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₅CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₇CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₉CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₄CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₆CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₈CH₂CH₂Si(OCH₃)₃;

CF₃(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₃(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₅(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₇(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₃CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₅CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₇CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₉CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₄CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₆CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₈CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₃(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₅(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₇(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₃CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₅CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₇CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₉CH₂CH₂Si(OCH₂CH₃)₃; and

CF₃(CF₂)₇SO₂N(C₂H₅)C₂H₄CH₂Si(OCH₃)₃.

Through the use of the above polysiloxane comprising a fluoroalkyl groupas a physicochemical cell adhesive material, a portion not subjected toenergy irradiation of a photocatalyst-comprising cell adhesivenessvariation layer will form a surface lacking cell adhesiveness because ofthe presence of a portion having fluorine on the surface. On the otherhand, a portion subjected to energy irradiation will form a surfacehaving cell adhesiveness because of the removal of fluorine and the likeand the resulting presence of a portion having an OH group and the likeon the surface. Therefore, regions can be patterned so that portionssubjected to energy irradiation and portions not subjected to energyirradiation differ in terms of cell adhesiveness.

Moreover, an example of reactive silicone in (2) above is a compoundhaving a backbone that is represented by the following general formula.

In the above formula, “n” is an integer of 2 or greater. R¹ and R² eachindicate a C₁₋₁₀ substituted or unsubstituted alkyl, alkenyl, or arylgroup. Examples of a substituent include halogen and cyano. Specificexamples of R¹ and R² include methyl, ethyl, propyl, vinyl, phenyl,phenyl halide, cyano methyl, cyano ethyl, and cyano propyl. Vinyl,phenyl, or phenyl halide preferably constitutes 40% or less (in molarratio) of the whole. Furthermore, a compound wherein R¹ and R² are eacha methyl group is preferable because this results in the lowest surfaceenergy. In addition, a methyl group constitutes preferably 60% (in molarratio) or more of the whole. Furthermore, a chain terminus or a sidechain has at least 1 or more reactive groups such as a hydroxyl group ina molecular chain.

Moreover, an organosilicone compound that does not undergo acrosslinking reaction and thus is stable, such as dimethyl polysiloxane,may also be separately mixed with the above organopolysiloxane.

Furthermore, an example of a physicochemical cell adhesive material (ofa decomposable substance type) is a surfactant that is decomposed by theaction of a photocatalyst and that has a function (exerted bydecomposition) of varying the polarity of the surface of aphotocatalyst-comprising polarity variation layer. Specific examples ofsuch surfactant include hydrocarbon non-ionic surfactants (e.g., NIKKOLBL, BC, BO, and BB series produced by Nikko Chemicals Co., Ltd.) andfluorine or silicone non-ionic surfactants (e.g., ZONYL FSN and FSOproduced by DuPont Kabushiki Kaisha, Surflon S-141 and 145 produced byASAHI GLASS CO., LTD., Megafac F-141 and 144 produced by DAINIPPON INKAND CHEMICALS, INCORPORATED, FTERGENT F-200 and F251 produced by NEOSCOMPANY LIMITED, Unidine DS-401 and 402 produced by DAIKIN INDUSTRIES,LTD., and Fluorad FC-170 and 176 produced by 3M (Minnesota Mining andManufacturing Company). Moreover, a cationic surfactant, an anionicsurfactant, or an amphoteric surfactant can also be used.

In addition, when a physicochemical cell adhesive material of adecomposable substance type is used as a material, generally preferablya binder component is separately used. Such binder component that isused in this case is not particularly limited, as long as it possessesbinding energy that is sufficiently high so that the main backbone isnot decomposed by the action of the above photocatalyst. Specificexamples of such component include polysiloxane having no organicsubstituent and polysiloxane having little organic substituents. Theycan also be obtained by hydrolysis and polycondensation oftetramethoxysilane, tetraethoxysilane, or the like.

In addition, in this embodiment, a physicochemical cell adhesivematerial of such binder type and a physicochemical cell adhesivematerial of such decomposable substance type may be used together.

Another example is a physicochemical cell adhesiveness variationmaterial whose cell adhesiveness is varied through the control ofelectrostatic interaction. In the case of such material, positivelycharged functional groups (contained in such material) are decomposed bythe action of a photocatalyst along with energy irradiation. The amountof positive charge existing on the surface is then varied so as to varyadhesiveness between the surface and cells. Thus, a cell adhesivenessvariation pattern is formed. An example of such material is poly Llysine.

b. Biological Cell Adhesiveness Variation Material

Examples of a biological factor for causing cells to adhere to thesurface include a material that can have a property of adhering to manycell types and a material that has a property of adhering to only aspecific cell type. The former material is a collagen I type material,for example. The latter material ispoly(N-p-vinylbenzyl[O-β-D-galactopyranosyl-(1→4)-D-gluconamide])(hereinafter,PVLA) that causes selective adhesion of hepatic parenchymal cells, forexample. In the case of PVLA, it is inferred that selective and specificmaterial-to-cell adhesion occurs, because PVLA has a galactose groupthat is specifically recognized by hepatic parenchymal cells in itsstructure.

When such material is mixed with a photocatalyst and then used for aphotocatalyst-comprising cell adhesiveness variation layer, thefollowing type of usage is possible. Collagen I type material issolubilized by enzyme treatment. The thus solubilized collagen I ismixed with a photocatalyst such as a TiO₂ particle that has beenpreviously subjected to calcination treatment and grinding treatment,thereby preparing a material for a photocatalyst-comprising celladhesiveness variation layer. Next, the material for thephotocatalyst-comprising cell adhesiveness variation layer is applied toa substrate, thereby forming a photocatalyst-comprising celladhesiveness variation layer. When the photocatalyst-comprising celladhesiveness variation layer is irradiated with a small amount ofenergy, a cell adhesive peptide structure in a side chain of collagen ispartially disrupted. Thus, cell adhesiveness can be decreased.Furthermore, such cell adhesive peptide structure can be graduallycaused to disappear by increasing the amount of energy irradiation.Furthermore, cell adhesiveness can be further decreased.

Furthermore, an excessive amount of energy irradiation can lead todisruption of the main chain structure of collagen and complete loss ofthe cell adhesiveness.

(2) Photocatalyst

Examples of a photocatalyst that is used in this embodiment includethose known as optical semiconductors such as titanium dioxide (TiO₂),zinc oxide (ZnO), tin oxide (SnO₂), strontium titanate (SrTiO₃),tungsten oxide (WO₃), bismuth oxide (Bi₂O₃), and iron oxide (Fe₂O₃). 1or 2 or more types of optical semiconductor can be selected from theabove examples, mixed, and then used.

In this embodiment, titanium dioxide is particularly preferably usedbecause it possesses high band gap energy, is chemically stable, is freefrom toxicity, and can be easily obtained. There exist anatase-type andrutile-type titanium dioxide, and both can be used in this embodiment.The anatase-type titanium dioxide is preferable. The anatase typetitanium dioxide has an excitation wavelength of 380 nm or less.

Examples of such anatase-type titanium dioxide include anatase-typetitaniasol of hydrochloric acid deflocculation type (STS-02 (averageparticle size of 7 nm) produced by ISHIHARA SANGYO KAISHA, LTD. andST-K01 produced by ISHIHARA SANGYO KAISHA, LTD.) and anatase-typetitaniasol of nitric acid deflocculation type (TA-15 (average particlesize of 12 nm) produced by NISSAN CHEMICAL INDUSTRIES, LTD.).

A smaller photocatalyst particle size is preferable, becausephotocatalyst reactions take place more effectively with smallerparticle size. The average particle diameter is preferably 50 nm orless. It is particularly preferable to use a photocatalyst with anaverage diameter of 20 nm or less.

The content of a photocatalyst in a photocatalyst-comprising celladhesiveness variation layer that is used in this embodiment can bedetermined to be within a range between 5 wt. % and 60 wt. %, andpreferably between 20 wt. % and 40 wt. %.

2. Substrate

A substrate that is used as the cell array substrate of the presentinvention is not particularly limited, as long as it is formed of amaterial with which a photocatalyst-comprising cell adhesivenessvariation layer can be formed on the surface. Any form can be selectedfor such substrate, as long as surface treatment can be carried outthrough exposure treatment. Specific examples of such material includeinorganic materials such as metal, glass, and silicon and organicmaterials represented by plastic. The shape of such substrate is alsonot limited. Examples of such shape include a flat plate, a flatmembrane, a film, and a porous membrane.

3. Cell Adhesiveness Variation Pattern

In this embodiment, the above-described photocatalyst—comprising celladhesiveness variation layer is formed on the above substrate, and thenthe substrate is subjected to energy irradiation in a pattern. Thus, acell adhesiveness variation pattern with variation in cell adhesivenessis formed.

Such cell adhesiveness variation pattern is generally formed of regionshaving good cell adhesiveness (with good cell adhesiveness) and regionshaving inhibited cell adhesiveness (with poor cell adhesiveness).Through adhesion of cells to the regions having good cell adhesiveness,the cells can be adhered in a high-definition pattern. Such regionshaving good cell adhesiveness and such regions having inhibited celladhesiveness are determined depending on the type of a cell adhesivenessvariation material that is used herein.

For example, a cell adhesiveness variation material may be aphysicochemical cell adhesiveness variation material that causesvariation in cell adhesiveness by varying the surface free energy. Insuch case, the surface free energy that is within a predetermined rangetends to result in good cell adhesiveness, but the surface free energythat is out of such range tends to result in decreased celladhesiveness. A known example of variation in cell adhesiveness due tosurface free energy is provided by the experimental results shown in thelower part of page 109, Frontiers of Biomaterials, under the generaleditorship of Yoshihito Ikada, CMC Publishing CO., LTD.

Cell adhesiveness can also be determined depending not only on thesurface free energy of the above material, but also on the combinationof a cell type and a material type that are caused to come into contact.

Here, such cell adhesiveness variation pattern is a pattern comprisingthe above regions having good cell adhesiveness and the above regionshaving inhibited cell adhesiveness. Depending on its application, it maybe a cell adhesiveness variation pattern comprising regions wheresurface cell adhesiveness is varied by at least 3 different levels.

This is because, in a case where a photocatalyst-comprising celladhesiveness variation layer comprising a biological cell adhesivenessvariation material is used or in a case where it has not yet beenconfirmed if cell adhesiveness is good, or the like, it may beadvantageous in terms of ability of finding optimum states foradhesiveness by successively causing changes to the surface states of aphotocatalyst-comprising cell adhesiveness variation layer.

As described above, in the present invention, 3 or more different levelsof cell adhesiveness include a state where cell adhesiveness issuccessively varied. The appropriate level of cell adhesiveness isappropriately selected depending on each circumstance and thendetermined.

Regions having such multiple different levels of adhesiveness can beformed by varying the amount of energy irradiation to aphotocatalyst-comprising cell adhesiveness variation layer. Specificexamples of such method include a method using half-tone photomasksvarying in transmittance and a method that involves performing overlapexposure more than once using a plurality of photomasks differing fromeach other in shielding portion pattern.

Furthermore, a cell adhesiveness variation pattern that can be used inthis embodiment uses a difference in photocatalyst activity between aportion subjected to energy irradiation and a portion not subjected toenergy irradiation. Specifically, for example, a biological celladhesiveness variation material that has been introduced as adecomposable substance into a photocatalyst-comprising cell adhesivenessvariation layer is used. When the surface of suchphotocatalyst-comprising cell adhesiveness variation layer is irradiatedwith energy in a pattern, the biological cell adhesiveness variationmaterial that has exuded on the surface of the irradiated portion isdecomposed and the biological cell adhesiveness variation material ofthe unirradiated portion remains. Hence, when such biological celladhesiveness variation material has good adhesiveness to a specific celltype or has good adhesiveness to many cell types, such unirradiatedportions become to be regions having good cell adhesiveness. Theirradiated portions become to be regions where a biological celladhesiveness variation material having good adhesiveness to a cell isabsent. Furthermore, the irradiated portions also become to be regionswhere a photocatalyst having activated sterility has been exposed as aresult of energy irradiation. Therefore, when an energy-irradiatedportion results in regions having inhibited cell adhesiveness,particularly when culture is carried out using the cell array substratein this embodiment for a predetermined time period, such regions areadvantageous in terms of not causing problems such as a wider patternwidth.

B. 2^(nd) Embodiment

A 2^(nd) embodiment of the cell array substrate of the present inventionis a cell array substrate comprising a substrate and a cell adhesivenessvariation layer that is formed on the substrate and comprises a celladhesiveness variation material whose cell adhesiveness is varied by theaction of a photocatalyst along with energy irradiation, wherein theabove cell adhesiveness variation layer forms a cell adhesivenessvariation pattern with variation in cell adhesiveness characterized inthat: the above cell adhesiveness variation layer comprises aphotocatalyst-comprising photocatalyst treatment layer and a celladhesiveness variation material layer that is formed on the abovephotocatalyst treatment layer and comprises the above cell adhesivenessvariation material.

In this embodiment, such cell adhesiveness variation layer comprises aphotocatalyst treatment layer formed on a substrate and a celladhesiveness variation material layer formed on the photocatalysttreatment layer. Thus, when energy irradiation is carried out, the celladhesiveness of the cell adhesiveness variation material within the celladhesiveness variation material layer is varied by the action of thephotocatalyst within the photocatalyst treatment layer. Hence, a celladhesiveness variation pattern comprising portions subjected to energyirradiation and portions not subjected to energy irradiation, where theportions differ in terms of cell adhesiveness, can be formed.

Members used in such cell array substrate in this embodiment will beseparately described as follows.

1. Cell Adhesiveness Variation Material Layer

In the cell array substrate of this embodiment, a cell adhesivenessvariation material layer is formed on a photocatalyst treatment layerthat is formed on the substrate. As such cell adhesiveness variationmaterial layer, a layer that is formed with the use of a celladhesiveness variation material explained in the above 1^(st) embodimentcan be used. A cell adhesiveness variation material layer prepared withthe use of a physicochemical cell adhesiveness variation material and acell adhesiveness variation material layer prepared with the use of abiological cell adhesiveness variation material will be separatelyexplained as follows.

(1) Use of Physicochemical Cell Adhesiveness Variation Material

In this embodiment, a cell adhesiveness variation material layer formedof a physicochemical cell adhesiveness variation material may beprepared as a layer prepared with the use of a material similar to thatexplained in the above 1^(st) embodiment. When such material is used,the thus prepared layer is similar to the above layer except for thepresence or the absence of a photocatalyst. In addition, in thisembodiment, a cell adhesiveness variation material layer is notprincipally required to comprise a photocatalyst therewithin, but maycomprise a photocatalyst in a small amount in view of sensitivity or thelike.

Furthermore, in this embodiment, a cell adhesiveness variation materiallayer is formed as a layer to be decomposed and removed (that is, thelayer is decomposed and removed by the action of a photocatalyst) on aphotocatalyst treatment layer. Regions wherein the cell adhesivenessvariation material layer has been decomposed by the action of thephotocatalyst along with energy irradiation (that is, regions whereinthe photocatalyst treatment layer has been exposed) and regions whereinthe cell adhesiveness variation material layer has remained are thenformed. Thus, a cell adhesiveness variation pattern is formed. Such typeof cell adhesiveness variation material layer having the thus formedpattern can be used.

Specifically, when cell adhesiveness is controlled with surface freeenergy, a physicochemical cell adhesiveness variation material whosesurface free energy is appropriate for cell adhesiveness is used. Suchmaterial is applied to the whole surface, thereby forming a celladhesiveness variation material layer. Subsequently, patterned energyirradiation is carried out according to a pattern, so as to form apattern comprising regions of presence of and of absence of the celladhesiveness variation material layer. Thus, a cell adhesivenessvariation pattern is formed.

Examples of such physicochemical cell adhesiveness variation materiallayer that is used as a layer to be decomposed and removed and can beused for controlling cell adhesiveness with surface free energy includeregenerated cellulose and nylon 11.

Furthermore, when cell adhesiveness is controlled with electrostaticinteraction, a cell adhesiveness variation pattern can be formed using apositively charged physicochemical cell adhesiveness variation materialand by a method similar to the above method.

Examples of a material that is used for such physicochemical celladhesiveness variation material layer used as a layer to be decomposedand removed and can be used for controlling cell adhesiveness withelectrostatic interaction include polyamine graftpoly(2-hydroxymethylmethacrylate)(HA-x) and the like.

These resins are dissolved in a solvent and a film can be formed by ageneral film production method such as a spin coat method. Moreover, inthe present invention, a defect-free film can be formed with the use ofa functional thin film such as a self-organizing monomolecular film,Langmuir-Blodgett film, or an alternate adsorption film. Thus, it ispreferable to use such film production method.

When a cell adhesiveness variation pattern is formed using a celladhesiveness variation material layer as such layer to be decomposed andremoved, regions subjected to decomposition and removal are regionswithin which cell culture is greatly inhibited, because in such regionsa photocatalyst treatment layer is exposed. Hence, a cell arraysubstrate that is obtained by such method is advantageous in that it canmaintain a high-definition pattern even after keeping cells for a longtime.

(2) Use of Biological Cell Adhesiveness Variation Material

In this embodiment, a material similar to that explained in the 1^(st)embodiment can be used for a cell adhesiveness variation material layerthat is formed of a biological cell adhesiveness variation material. Anexample of such material is collagen I type.

2. Photocatalyst Treatment Layer

Next, a photocatalyst treatment layer that is used in the presentinvention will be explained. Such photocatalyst treatment layer used inthe present invention is not particularly limited, as long as it isconstituted in such a manner that the cell adhesiveness of a celladhesiveness variation material layer (which is formed on aphotocatalyst treatment layer) is varied by a photocatalyst in thephotocatalyst treatment layer. Such photocatalyst treatment layer may bea layer composed of a photocatalyst and a binder or a layer prepared bya film production method using a photocatalyst alone. Furthermore, thesurface may particularly be lyophilic or lyophobic. A lyophilic surfaceis preferable in terms of convenience of forming a cell adhesivenessvariation material layer and the like on the photocatalyst treatmentlayer.

In such photocatalyst treatment layer, the action mechanism of aphotocatalyst represented by titanium oxide that is described later isnot always clear. It is thought that a direct reaction between a carriergenerated by light irradiation and a neighboring compound or activeoxygen species generated in the presence of oxygen and water causes achange in the chemical structure of organic matters. In the presentinvention, it is considered that such carrier acts on a compound in acell adhesiveness variation material layer formed on a photocatalysttreatment layer. Examples of such photocatalyst are similar to thosedescribed in detail in the 1^(st) embodiment.

The photocatalyst treatment layer in this embodiment may be a layerformed of a photocatalyst alone as described above or a layer formed bymixing it with a binder.

A photocatalyst treatment layer consisting of a photocatalyst alone isadvantageous in terms of cost, because its efficiency of causingvariation in the cell adhesiveness of a cell adhesiveness variationmaterial layer is improved and a treatment time is reduced. On the otherhand, a photocatalyst treatment layer consisting of a photocatalyst anda binder is advantageous in that a photocatalyst treatment layer can beeasily formed.

Examples of a method for forming a photocatalyst treatment layerconsisting of a photocatalyst alone include a sputtering method, a CVDmethod, and a vacuum film production method such as a vacuum depositionmethod. Formation of a photocatalyst treatment layer by the vacuum filmproduction method enables preparation of a photocatalyst treatment layerformed of a uniform film and comprising a photocatalyst alone. Thus, theproperties of a cell adhesiveness variation material layer can beuniformly varied. Furthermore, since such layer consists of aphotocatalyst alone, it becomes possible to vary the cell adhesivenessof a cell adhesiveness variation layer more efficiently than in the caseof using a binder.

Moreover, another example of a method for forming a photocatalysttreatment layer consisting of a photocatalyst alone is, when aphotocatalyst is titanium dioxide, a method that comprises formingamorphous titania on a substrate and then causing a phase change throughcalcination to obtain crystalline titania. Amorphous titania that isused herein can be obtained by hydrolysis or dehydration andcondensation of inorganic salts of titanium, such as titaniumtetrachloride and titanium sulfate, or hydrolysis, or dehydration andcondensation of an organic titanium compound, such as tetraethoxytitanium, tetraisopropoxy titanium, tetra-n-propoxy titanium,tetrabutoxy titanium, and tetramethoxy titanium in the presence of acid.Subsequently, such titania is modified to result in anatase-type titaniathrough calcination at a temperature between 400° C. and 500° C. or toresult in rutile-type titania through calcination at a temperaturebetween 600° C. and 700° C.

Furthermore, when a binder is used, a preferable binder possessesbinding energy that is sufficiently high so that the main backbone ofthe binder is not decomposed by the action of the above photocatalyst.Examples of such binder include the above organopolysiloxane and thelike.

When organopolysiloxane is used as a binder as described above, theabove photocatalyst treatment layer can be formed by dispersing aphotocatalyst and organopolysiloxane that is the binder in a solventtogether with another additive, if necessary, preparing an applicationsolution, and then applying the application solution to a transparentsubstrate. A solvent that is used herein is preferably an alcohol-basedorganic solvent such as ethanol or isopropanol. Application can becarried out by a known application method such as a spin coating, aspray coating, a dip coating, a roll coating, or a bead coating method.When a UV-hardened type component is contained as a binder, aphotocatalyst treatment layer can be formed through UV irradiation tocarry out hardening treatment.

Furthermore, an amorphous silica precursor can be used as a binder. Assuch amorphous silica precursor, a silicon compound represented bygeneral formula SiX₄, wherein X is halogen, a methoxy group, an ethoxygroup, an acetyl group, or the like, silanol that is a hydrolysatethereof, or polysiloxane with an average molecular weight of 3000 orless is preferable.

Specific examples of such precursor include tetraethoxysilane,tetraisopropoxy silane, tetra-n-propoxy silane, tetrabutoxy silane, andtetramethoxysilane. Furthermore, in this case, a photocatalyst treatmentlayer can be formed by uniformly dispersing an amorphous silicaprecursor and photocatalyst particles in a non-aqueous solvent,subjecting the resultant to hydrolysis on a transparent substrate bywater in air, so as to form silanol, and then carrying out dehydration,condensation, and polymerization at normal temperature. A process ofdehydration, condensation, and polymerization of silanol at 100° C. orhigher results in an increased polymerization degree of silanol and thuscan improve the strength of the film surface. Moreover, such bindingagent can be used alone, or a mixture of 2 or more types of such bindingagents can be used.

The content of a photocatalyst in a photocatalyst treatment layer when abinder is used can be determined within a range between 5 wt. % and 60wt. %, and preferably between 20 wt. % and 40 wt. %. Furthermore, thethickness of a photocatalyst treatment layer is preferably within arange between 0.05 μm and 10 μm.

Furthermore, a photocatalyst treatment layer may comprise a surfactantin addition to the above photocatalyst and binder. Specific examples ofsuch surfactant include hydrocarbon non-ionic surfactants (e.g., NIKKOLBL, BC, BO, and BB series produced by Nikko Chemicals Co., Ltd.),fluorine or silicone non-ionic surfactants (e.g., ZONYL FSN and FSOproduced by DuPont Kabushiki Kaisha, Surflon S-141 and 145 produced byASAHI GLASS CO., LTD., Megafac F-141 and 144 produced by DAINIPPON INKAND CHEMICALS, INCORPORATED, FTERGENT F-200 and F251 produced by NEOSCOMPANY LIMITED, Unidine DS-401 and 402 produced by DAIKIN INDUSTRIES,LTD., and Fluorad FC-170 and 176 produced by 3M (Minnesota Mining andManufacturing Company). Moreover, a cationic surfactant, an anionicsurfactant, or an amphoteric surfactant can also be used.

Furthermore, a photocatalyst treatment layer can comprise, in additionto the above surfactant, an oligomer, a polymer, or the like such aspolyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene,diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenolresin, polyurethane, melamine resin, polycarbonate, poly(vinylchloride), polyamide, polyimide, styrene butadiene rubber,chloroprene-rubber, polypropylene, polybutylene, polystyrene, poly(vinylacetate), polyester, polybudadiene, polybenzimidazole, polyacrylnitrile, epichlorohydrin, polysulfide, and polyisoprene.

3. Substrate

A substrate that is used in this embodiment is not particularly limited,as long as the above photocatalyst treatment layer can be formed. Asubstrate similar to that explained in the 1^(st) embodiment can beused.

4. Cell Adhesiveness Variation Pattern

In this embodiment, a cell adhesiveness variation pattern is formed,wherein the cell adhesiveness of the surface of a cell adhesivenessvariation material layer is varied by the action of a photocatalyst in aphotocatalyst treatment layer as a result of energy irradiation carriedout in a pattern on the above cell adhesiveness variation materiallayer.

C. Third Embodiment

A cell array substrate in this embodiment comprises a substrate and acell adhesiveness variation layer that is formed on the above substrateand comprises a cell adhesiveness variation material whose celladhesiveness is varied by the action of a photocatalyst along withenergy irradiation, wherein the above cell adhesiveness variation layerforms a cell adhesiveness variation pattern with variation in celladhesiveness, characterized in that: the above cell adhesivenessvariation layer is a cell adhesiveness variation material layercomprising the above cell adhesiveness variation material; and the aboveadhesiveness variation pattern is formed by arranging aphotocatalyst-comprising layer and the above cell adhesiveness variationmaterial layer so that the layers face each other, and then carrying outenergy irradiation from a predetermined direction.

In this embodiment, a cell adhesiveness variation layer is a celladhesiveness variation material layer as described above; and the aboveadhesiveness variation pattern is formed by arranging aphotocatalyst-comprising layer and the above cell adhesiveness variationmaterial layer so that the layers face each other, and then carrying outenergy irradiation from a predetermined direction. Thus, at the time ofenergy irradiation, the cell adhesiveness of the cell adhesivenessvariation material within the cell adhesiveness variation material layeris varied by the action of the photocatalyst within thephotocatalyst-comprising layer. Hence, such cell adhesiveness variationpattern can be formed, comprising portions subjected to energyirradiation and portions not subjected to energy irradiation, where suchportions differ in terms of cell adhesiveness.

Members used in such cell array substrate in this embodiment will beseparately explained.

1. Cell Adhesiveness Variation Material Layer

In the case of a cell array substrate of this embodiment, a celladhesiveness variation material layer is formed on the substrate. Suchcell adhesiveness variation material layer is similar to a layer that isformed with the use of a material explained in the above 2^(nd)embodiment. In addition, in this embodiment, a cell adhesivenessvariation material layer is not principally required to comprise aphotocatalyst therewithin, but may comprise a photocatalyst in a smallamount in view of sensitivity and the like.

Furthermore, in this embodiment, in a manner similar to that in theabove 2^(nd) embodiment, a cell adhesiveness variation material layermay be formed as a layer to be decomposed and removed (that is, thelayer is subjected to decomposition and removal through the action of aphotocatalyst) on a substrate. In this case, energy irradiation iscarried out using a photocatalyst-comprising-layer-side base plate, soas to form regions wherein the cell adhesiveness variation materiallayer has been decomposed by the action of the photocatalyst along withenergy irradiation (that is, the regions where the substrate has beenexposed) and regions where the cell adhesiveness variation materiallayer has remained. Thus, a cell adhesiveness variation pattern isformed. Such type of cell adhesiveness variation material layer havingthe thus formed pattern is used.

2. Substrate

A substrate that is used in this embodiment is not particularly limited,as long as the above cell adhesiveness variation material layer can beformed. A substrate similar to that explained in the 1^(st) embodimentcan be used.

3. Photocatalyst-Comprising Layer

Next, a photocatalyst-comprising layer that is used in this embodimentwill be explained as follows. Such photocatalyst-comprising layer thatis used in this embodiment is a layer comprising a photocatalyst. Suchlayer is generally formed on a base body such as glass and then used. Inthis embodiment, such photocatalyst-comprising layer is arranged so thatthe layer and the above cell adhesiveness variation material layer faceeach other. Through energy irradiation, the cell adhesiveness of thecell adhesiveness variation material layer can be varied by the actionof the photocatalyst contained in such photocatalyst-comprising layer.In this embodiment, such photocatalyst-comprising layer is arranged at apredetermined position when energy irradiation is carried out, so that acell adhesiveness variation pattern can be formed. Thus, there is noneed to cause the above cell adhesiveness variation material layer tocomprise a photocatalyst. Hence, such photocatalyst-comprising layer isadvantageous in that a cell adhesiveness variation material layer can bekept free from the action of a photocatalyst over time.

Such photocatalyst-comprising layer is similar to the photocatalysttreatment layer that is explained in the above 2^(nd) embodiment.

4. Cell Adhesiveness Variation Pattern

In this embodiment, a cell adhesiveness variation pattern is formed inthe above cell adhesiveness variation material layer. Such celladhesiveness variation pattern is formed by carrying out energyirradiation in a pattern using the above photocatalyst-comprising layer,so as to vary the cell adhesiveness of the surface of the celladhesiveness variation material layer by the action of the photocatalystin the photocatalyst-comprising layer.

II. Method for Producing a Cell Array Substrate

Next, the method for producing a cell array substrate of the presentinvention will be explained. Examples of such method for producing thecell array substrate of the present invention include three embodimentsas described above. All of these embodiments are characterized by theformation of a substrate for pattern formation that comprises asubstrate and a layer formed on the substrate, whose adhesiveness can bevaried by the action of a photocatalyst along with energy irradiation,irradiating energy to the substrate for pattern formation, so as tocause the photocatalyst to act thereon, followed by the formation of acell adhesiveness variation pattern with variation in cell adhesiveness.

According to the method for producing the cell array substrate of thepresent invention, a layer whose cell adhesiveness is varied by theaction of a photocatalyst along with the above energy irradiation isformed. Thus, through energy irradiation in a required pattern, itbecomes possible to easily produce a cell array substrate on which acell adhesiveness variation pattern (with variation in cell adhesivenessin a high-definition pattern) is formed. Therefore, a cell arraysubstrate with a high-definition pattern can be produced with convenientsteps without using any treatment solution that adversely affects cells.Moreover, such production method does not require any modification of acell adhesiveness variation material. Thus, options for materialselection can be expanded. Furthermore, a biological cell adhesivenessvariation material that exerts specific adhesiveness described later canalso be used without any problems.

The above 1^(st) to 3^(rd) embodiments for the method for producing acell array substrate of the present invention will be separatelyexplained as follows.

A. 1^(st) Embodiment

First, the 1^(st) embodiment of the cell array substrate of the presentinvention will be explained. The 1^(st) embodiment of the method forproducing a cell array substrate of the present invention comprises: astep of forming a substrate for pattern formation that comprises asubstrate and a photocatalyst-comprising cell adhesiveness variationlayer formed on the above substrate and comprising a photocatalyst and acell adhesiveness variation material whose cell adhesiveness is variedby the action of the photocatalyst along with energy irradiation; and astep of forming a cell adhesiveness variation pattern by irradiatingenergy to the above photocatalyst-comprising cell adhesiveness variationlayer so as to vary the cell adhesiveness of the abovephotocatalyst-comprising cell adhesiveness variation layer.

A method for producing a cell array substrate in this embodiment iscarried out as shown in FIG. 1, for example. Specifically, a substratefor pattern formation 3 (the step of forming a substrate for patternformation (FIG. 1( a)) comprising a substrate 1 and aphotocatalyst-comprising cell adhesiveness variation layer 2 formed onthe substrate 1 is formed. Next, the step of forming a cell adhesivenessvariation pattern (FIG. 1( c)) is carried out by irradiating the abovephotocatalyst-comprising cell adhesiveness variation layer 2 with energy5 using a photomask 4, for example (FIG. 1( b)), and then forming a celladhesiveness variation pattern 6 wherein the cell adhesiveness of aphotocatalyst-comprising cell adhesiveness variation layer 2 has beenvaried.

In this embodiment, a photocatalyst-comprising cell adhesivenessvariation layer comprising a photocatalyst and the above celladhesiveness variation material is formed. Through energy irradiation inthe step of forming a cell adhesiveness variation pattern, the celladhesiveness of the cell adhesiveness variation material is varied bythe action of the photocatalyst within the photocatalyst-comprising celladhesiveness variation layer. Thus a cell adhesiveness variation patterncomprising portions subjected to energy irradiation and portions notsubjected to energy irradiation, where the portions differ in terms ofcell adhesiveness, can be formed. Each step of this embodiment will beexplained.

1. Step of Forming a Substrate for Pattern Formation

First, the step of forming a substrate for pattern formation in thisembodiment will be explained. The step of forming a substrate forpattern formation in this embodiment is a step for forming suchsubstrate that comprises a substrate and a photocatalyst-comprising celladhesiveness variation layer that is formed on the substrate andcomprises a photocatalyst and a cell adhesiveness variation materialwhose cell adhesiveness is varied by the action of the photocatalystalong with energy irradiation.

This step can be carried out by applying a coating solution comprising aphotocatalyst and a cell adhesiveness variation material to a substrateby a known application method such as a spin coating, a spray coating, adip coating, a roll coating, or a bead coating method and thus forming aphotocatalyst-comprising cell adhesiveness variation layer. When aUV-hardened type component is contained as a binder, aphotocatalyst-comprising layer can be formed through UV irradiation tocarry out hardening treatment.

2. Step of Forming a Cell Adhesiveness Variation Pattern

Next, the step of forming a cell adhesiveness variation pattern in thisembodiment will be explained. The step of forming a cell adhesivenessvariation pattern in this embodiment is a step for forming such patternby subjecting the above photocatalyst-comprising cell adhesivenessvariation layer to energy irradiation and thus forming a celladhesiveness variation pattern wherein the cell adhesiveness of theabove photocatalyst-comprising cell adhesiveness variation layer hasbeen varied.

With this step, wherein energy irradiation is carried out in a desiredpattern, the cell adhesiveness of only the regions (of aphotocatalyst-comprising cell adhesiveness variation layer) subjected toenergy irradiation can be varied. Furthermore, a high-definition celladhesiveness variation pattern comprising regions having good celladhesiveness and regions having poor cell adhesiveness can be formed.

“Energy irradiation (exposure)” used in this embodiment is a conceptthat includes any form of irradiation with energy rays capable ofcausing variation in the cell adhesiveness on the surface of aphotocatalyst-comprising cell adhesiveness variation layer. Such energyirradiation is not limited to irradiation with visible light.

Light wavelength that is used for such energy irradiation is generallydetermined to be 400 nm or less and preferably 380 nm or less. This isbecause a preferable photocatalyst that is used for aphotocatalyst-comprising cell adhesiveness variation layer as describedabove is titanium dioxide and light having a wavelength as describedabove is preferable as energy to activate the action of a photocatalystwith the use of such titanium dioxide.

Examples of a light source that can be used for such energy irradiationinclude a mercury lamp, a metal halide lamp, a xenon lamp, an excimerlamp, and other various light sources.

In addition to a method that comprises carrying out patternedirradiation via a photomask using the above light source, a method thatcomprises carrying out irradiation so as to draw a pattern using a lasersuch as excimer or YAG can also be used.

The amount of energy irradiation should be the amount of irradiationrequired for varying the cell adhesiveness of the surface of aphotocatalyst-comprising cell adhesiveness variation layer by the actionof the photocatalyst within such layer.

The cell adhesiveness of the surface of a photocatalyst-comprising celladhesiveness variation layer is varied depending on the amount of energyirradiation. Hence, the adhesiveness can be adjusted by regulating theenergy irradiation time, for example. Therefore, the surface can beprepared to have proper adhesiveness. Cell adhesiveness can be evaluatedusing the water contact angle on the surface as described above. Throughregulation of the energy irradiation time to obtain a surface having aproper water contact angle, a surface having a proper adhesiveness canbe prepared. For example, when fluoroalkyl silane is used as a celladhesiveness variation material and the material is irradiated withultraviolet light at 365 nm and at an intensity of 25.0 mW/second, andthen quartz is used for the substrate of a photomask, a surface havingpreferable adhesiveness can be prepared through generally 120 to 600seconds and preferably 240 to 480 seconds of irradiation. Such energyirradiation time, irradiation intensity, and the like can beappropriately regulated depending on a material for a substrate, a celladhesiveness variation material, and the like to be used herein.

At this time, it is preferable to carry out energy irradiation whileheating a photocatalyst-comprising cell adhesiveness variation layer.This is preferable because sensitivity can be elevated and celladhesiveness can be varied efficiently. Specifically, heating within arange between 30° C. and 80° C. is preferable.

Regarding direction for energy irradiation in this embodiment, when theabove substrate is transparent, patterned energy irradiation or laserirradiation to draw a pattern can also be carried out via a photomaskfrom either the substrate side or the photocatalyst-comprising celladhesiveness variation layer side. On the other hand, when a substrateis opaque, energy irradiation should be carried out from thephotocatalyst-comprising cell adhesiveness variation layer side.

B. 2^(nd) Embodiment

Next, the 2^(nd) embodiment of the method for producing a cell arraysubstrate of the present invention will be explained. The 2^(nd)embodiment of the cell array substrate of the present inventioncomprises: a step of forming a substrate for pattern formation thatcomprises a substrate, a photocatalyst-comprising photocatalysttreatment layer formed on the above substrate, and a cell adhesivenessvariation material layer being formed on the above photocatalysttreatment layer and comprising a cell adhesiveness variation materialwhose cell adhesiveness is varied by the action of the photocatalystalong with energy irradiation; and a step of forming a cell adhesivenessvariation pattern by subjecting the above cell adhesiveness variationmaterial layer to energy irradiation so as to vary the cell adhesivenessof the above cell adhesiveness variation material layer.

The method for producing a cell array substrate in this embodiment iscarried out as shown in FIG. 2, for example. Specifically, a substratefor pattern formation 3 (the step of forming a substrate for patternformation (FIG. 2( a)) comprising a substrate 1, a photocatalysttreatment layer 7 that is formed on the substrate 1, and a celladhesiveness variation material layer 8 that is formed on thephotocatalyst treatment layer 7 is formed. Next, the step of forming acell adhesiveness variation pattern (FIG. 2( c)) is carried out byirradiating the above cell adhesiveness variation material layer 8 withenergy 5 using a photomask 4, for example (FIG. 2( b)), and then forminga cell adhesiveness variation pattern 6 wherein the cell adhesiveness ofthe cell adhesiveness variation material layer 8 has been varied.

In this embodiment, a photocatalyst treatment layer and the above celladhesiveness variation material layer are formed. Through energyirradiation in the step of forming a cell adhesiveness variationpattern, the cell adhesiveness within the cell adhesiveness variationmaterial layer is varied by the action of the photocatalyst contained inthe photocatalyst treatment layer. Thus, a cell adhesiveness variationpattern comprising portions subjected to energy irradiation and portionsnot subjected to energy irradiation, where the portions differ in termsof cell adhesiveness, can be formed. Each step of this embodiment willbe explained as follows.

1. Step of Forming a Substrate for Pattern Formation

First, the step of forming a substrate for pattern formation in thisembodiment will be explained. The step of forming a substrate forpattern formation in this embodiment is a step for forming suchsubstrate that comprises a photocatalyst-comprising photocatalysttreatment layer formed on the above substrate and a cell adhesivenessvariation material layer that is formed on the above photocatalysttreatment layer and comprises a cell adhesiveness variation materialwhose cell adhesiveness is varied by the action of the photoctalystalong with energy irradiation.

Such photocatalyst treatment layer that is formed in this step mayconsist of a photocatalyst alone or may be formed by mixture with abinder.

Examples of a method for forming a photocatalyst treatment layerconsisting of a photocatalyst alone include a sputtering method, a CVDmethod, and a vacuum film production method such as a vacuum depositionmethod. For example, a method that is used when a photocatalyst istitanium dioxide comprises forming amorphous titania on a substrate andthen causing a phase change through calcination to obtain crystallinetitania. Formation of a photocatalyst treatment layer by the vacuum filmproduction method enables preparation of a photocatalyst treatment layerformed of a uniform film and comprising a photocatalyst alone. Thus, thecell adhesiveness on a cell adhesiveness variation material layer can beuniformly varied. Furthermore, such layer consists of a photocatalystalone. Thus, it becomes possible to vary the cell adhesiveness on a celladhesiveness variation material layer more efficiently than in the caseof using a binder.

Furthermore, when a photocatalyst treatment layer is prepared by mixinga photocatalyst with a binder, such layer can be formed by preparing anapplication solution by dispersing such photocatalyst and such binder ina solvent together with another additive if necessary and then applyingthe thus prepared application solution to a transparent substrate. Asolvent that is used herein is preferably an alcohol-based organicsolvent such as ethanol or isopropanol. Application can be carried outby a known application method such as a spin coating, a spray coating, adip coating, a roll coating, or a bead coating method. When aUV-hardened type component is contained as a binder, a photocatalysttreatment layer can be formed through UV irradiation to carry outhardening treatment.

Subsequently, a coating solution comprising the above cell adhesivenessvariation material is applied onto the above photocatalyst treatmentlayer by a known application method such as a spin coating, a spraycoating, a dip coating, a roll coating, or a bead coating method. Thus,a cell adhesiveness variation material layer can be formed. When aUV-hardened type component is contained as a binder, a photocatalysttreatment layer can be formed through UV irradiation to carry outhardening treatment.

Such substrate, such photocatalyst treatment layer, and such celladhesiveness variation material layer that are used in this step aresimilar to those explained in the above section of the 2^(nd)embodiment, “I. Cell array substrate.”

2. Step of Forming a Cell Adhesiveness Variation Pattern

Next, the step of forming a cell adhesiveness variation pattern in thisembodiment will be explained. The step of forming a cell adhesivenessvariation pattern in this embodiment is a step for forming such patternby subjecting the above cell adhesiveness variation material layer toenergy irradiation and thus forming a cell adhesiveness variationpattern wherein the cell adhesiveness of the above cell adhesivenessvariation material layer has been varied.

With this step wherein energy irradiation is carried out in a desiredpattern, the cell adhesiveness of regions (of a cell adhesivenessvariation material layer) subjected to energy irradiation can beexclusively varied. Furthermore, a high-definition cell adhesivenessvariation pattern can be formed, which comprises regions having goodcell adhesiveness and regions having poor cell adhesiveness.

An energy irradiation method, energy to be used for irradiation, and theamount of energy irradiation that are used in this step are similar tothose in the above 1^(st) embodiment.

C. 3^(rd) Embodiment

Next, the 3^(rd) embodiment of the cell array substrate of the presentinvention will be explained. The 3^(rd) embodiment of the method forproducing a cell array substrate of the present invention comprises: astep of forming a substrate for pattern formation that comprises asubstrate and a cell adhesiveness variation material layer that isformed on the substrate and comprises a cell adhesiveness variationmaterial whose cell adhesiveness is varied by the action of aphotocatalyst along with energy irradiation; and a step of forming acell adhesiveness variation pattern by arranging the above substrate forpattern formation and a photocatalyst-comprising-layer-side base platethat comprises a photocatalyst-comprising layer and a base body so thatthe above cell adhesiveness variation material layer and the abovephotocatalyst-comprising layer face each other, carrying out energyirradiation from a predetermined direction, and thus forming a celladhesiveness variation pattern wherein the cell adhesiveness of theabove cell adhesiveness variation material layer has been varied.

The method for producing a cell array substrate in this embodiment iscarried out as shown in FIG. 3, for example. Specifically, a substratefor pattern formation 3 is formed (the step of forming a substrate forpattern formation (FIG. 3( a)), which comprises a substrate 1 and a celladhesiveness variation material layer 8 formed on the substrate 1. Next,a photocatalyst-comprising-layer-side base plate 13 is prepared, whichcomprises a base body 11 and a photocatalyst-comprising layer 12 formedon the base body 11. The step of forming a cell adhesiveness variationpattern is carried out (FIG. 3( c)) by arranging thephotocatalyst-comprising layer 12 in thephotocatalyst-comprising-layer-side base plate 13 and the above celladhesiveness variation material layer 8 so that the layers face eachother, irradiating with energy 5 using a photomask 4, for example (FIG.3( b)), and then forming a cell adhesiveness variation pattern 6 whereinthe cell adhesiveness of the cell adhesiveness variation material layer8 has been varied.

In this embodiment, the above cell adhesiveness variation material layeris formed. Through energy irradiation using the photocatalyst-comprisinglayer-side base plate in the step of forming a cell adhesivenessvariation pattern, the cell adhesiveness within the cell adhesivenessvariation material layer is varied by the action of the photocatalystcontained in the photocatalyst-comprising layer. Thus a celladhesiveness variation pattern can be formed, which comprises portionssubjected to energy irradiation and portions not subjected to energyirradiation, where the portions differ in terms of cell adhesiveness.Each step of this embodiment will be explained.

1. Step of Forming a Substrate for Pattern Formation

First, the step of forming a substrate for pattern formation in thepresent invention will be explained. The step of forming a substrate forpattern formation in the present invention is a step for forming suchsubstrate that comprises a substrate and a cell adhesiveness variationmaterial layer that is formed on the substrate and comprises a celladhesiveness variation material whose cell adhesiveness is varied by theaction of a photocatalyst along with energy irradiation.

This step can be carried out by applying a coating solution comprising acell adhesiveness variation material to a substrate by a knownapplication method such as a spin coating, a spray coating, a dipcoating, a roll coating, or a bead coating method and thus forming acell adhesiveness variation material layer. When a UV-hardened typecomponent is contained as a binder, a photocatalyst-comprising layer canbe formed through UV irradiation to carry out hardening treatment.

Such substrate and such cell adhesiveness variation material that can beused in this step are similar to those explained in the above section ofthe 1^(st) embodiment, “I. Cell array substrate.”

2. Step of Forming a Cell Adhesiveness Variation Pattern

Next, the step of forming a cell adhesiveness variation pattern in thisembodiment will be explained. The step of forming an adhesivenessvariation pattern in this embodiment is a step for forming such patternby arranging the above substrate for pattern formation and aphotocatalyst-comprising-layer-side base plate that comprises aphotocatalyst-comprising layer and a base body so that the above celladhesiveness variation material layer and the abovephotocatalyst-comprising layer face each other, and then carrying outenergy irradiation from a predetermined direction, so as to form apattern wherein the cell adhesiveness of a cell adhesiveness variationmaterial layer has been varied.

With this step wherein a photocatalyst-comprising layer in aphotocatalyst-comprising-layer-side base plate and a cell adhesivenessvariation material layer are arranged so that the layers face each otherand energy irradiation is carried out in a desired pattern, the celladhesiveness of regions (of a cell adhesiveness variation materiallayer) subjected to energy irradiation can be exclusively varied.Furthermore, a high-definition cell adhesiveness variation pattern canbe formed, which comprises regions having good cell adhesiveness andregions having poor cell adhesiveness.

Such photocatalyst-comprising-layer-side base plate and energyirradiation that are used and carried out, respectively, in this stepwill be separately explained as follows.

(1) Photocatalyst-Comprising-Layer-Side Base Plate

First, a photocatalyst-comprising-layer-side base plate that is used inthis embodiment will be explained.

Such photocatalyst-comprising-layer-side base plate that is used in thisembodiment comprises at least a photocatalyst-comprising layer and abase body. Such base plate is generally prepared by forming aphotocatalyst-comprising layer in the shape of thin film (formed by apredetermined method) on a base body. Furthermore, aphotocatalyst-comprising-layer-side base plate that can also be usedherein may comprise patterned photocatalyst-comprising-layer-sideshielding portions or a primer layer formed thereon.

In this embodiment, at the time of energy irradiation, the above celladhesiveness variation material layer and the photocatalyst-comprisinglayer in the above photocatalyst-comprising-layer-side base plate arearranged so that the layers face each other with a predetermined spacebetween the two. The cell adhesiveness of the cell adhesivenessvariation material layer is varied by the action of thephotocatalyst-comprising layer of thephotocatalyst-comprising-layer-side base plate. Thephotocatalyst-comprising-layer-side base plate is removed after energyirradiation, so that a cell adhesiveness variation pattern is formed.Each component of such photocatalyst-comprising-layer-side base platewill be explained.

a. Photocatalyst-Comprising Layer

A photocatalyst-comprising layer that is used in this embodimentcomprises at least a photocatalyst and may or may not comprise a binder.The photocatalyst-comprising layer is similar to the photocatalysttreatment layer described in the above 2^(nd) embodiment.

Such photocatalyst-comprising layer that is used in this embodiment maybe, as shown in FIG. 3, for example, a layer formed on the whole surfaceof a base body 11. For example, as shown in FIG. 4, thephotocatalyst-comprising layer may be a photocatalyst-comprising layer12 patterned on the base body 11.

By patterning of such photocatalyst-comprising layer, patternedirradiation using a photomask or the like is not required at the time ofenergy irradiation. Furthermore, through irradiation of the wholesurface, a cell adhesiveness variation pattern can be formed in a celladhesiveness variation material layer.

A patterning method for such photocatalyst-comprising layer is notparticularly limited. For example, such patterning can be carried out bya photolithography method or the like.

Furthermore, energy irradiation is carried out while closely contactinga photocatalyst-comprising layer and a cell adhesiveness variationmaterial layer with each other. In such case, the properties of onlyportions where the photocatalyst-comprising layer has been actuallyformed are varied. Thus, such case is advantageous in that energyirradiation may be carried out from any direction, as long as portionswhere the above photocatalyst-comprising layer and cell adhesivenessvariation material layer face each other are irradiated with energy.Another advantage is that energy to be used for irradiation is also notparticularly limited to parallel energy such as parallel light.

b. Base Body

In this embodiment, as shown in FIG. 3, thephotocatalyst-comprising-layer-side base plate 13 comprises at least abase body 11 and the photocatalyst-comprising layer 12 formed on thebase body 11. At this time, the material composing a base body usedherein is appropriately selected depending on the direction of energyirradiation described later, necessity for the transparency of the thusobtained cell array substrate, and the like.

Furthermore, such base body that is used in this embodiment may be abase body having flexibility, such as a film made of a resin, or a basebody lacking flexibility, such as a glass substrate. Moreover, asanother type of base body, an optical waveguide such as optical fibercan also be used. Such base body can be appropriately selected dependingon the energy irradiation method.

In addition, to improve close contact between the surface of a base bodyand a photocatalyst-comprising layer, an anchor layer may also be formedon the base body. Examples of such anchor layer include a silanecoupling agent and a titanium coupling agent.

c. Photocatalyst-Comprising-Layer-Side Shielding Portion

A photocatalyst-comprising-layer-side base plate that is used in thisembodiment may comprise photocatalyst-comprising-layer-side shieldingportions patterned thereon. With the use of suchphotocatalyst-comprising-layer-side base plate comprising thephotocatalyst-comprising-layer-side shielding portions, it is notrequired to use a photomask or to carry out laser irradiation to draw apattern at the time of energy irradiation. Furthermore, it is notrequired to carry out positioning for aphotocatalyst-comprising-layer-side base plate and a photomask. Hence, aconvenient step can be realized and no expensive apparatuses are neededfor drawing irradiation. Thus, the use of suchphotocatalyst-comprising-layer-side base plate is advantageous in termsof cost.

The following two embodiments are possible for suchphotocatalyst-comprising-layer-side base plate comprising suchphotocatalyst-comprising-layer-side shielding portions, depending on thepositions at which the photocatalyst-comprising-layer-side shieldingportions are formed.

One embodiment of the photocatalyst-comprising-layer-side base plate isprepared as shown in FIG. 5, for example, whereinphotocatalyst-comprising-layer-side shielding portions 14 are formed onthe base body 11 and the photocatalyst-comprising layer 12 is formed onthe photocatalyst-comprising-layer-side shielding portions 14. The otherembodiment of the photocatalyst-comprising-layer-side base plate isprepared as shown in FIG. 6, for example, wherein thephotocatalyst-comprising layer 12 is formed on the base body 11 and thephotocatalyst-comprising-layer-side shielding portions 14 are formed onthe photocatalyst-comprising layer 12.

In both embodiments, compared with the case of using a photomask,photocatalyst-comprising-layer-side shielding portions are arranged inthe vicinity of portions where the above photocatalyst-comprising layerand cell adhesiveness variation material layer are to be arranged. Thus,the effect of energy scattering within a base body and the like can bereduced. Thus, it becomes possible to carry out patterned energyirradiation in an extremely precise manner.

Furthermore, in the above embodiment wherephotocatalyst-comprising-layer-side shielding portions are formed on aphotocatalyst-comprising layer, when a photocatalyst-comprising layerand a cell adhesiveness variation material layer are arranged atpredetermined positions, the film thickness of eachphotocatalyst-comprising-layer-side shielding portion is prepared to bethe same as the width of the space between the two layers. Hence, theembodiment is advantageous in that the abovephotocatalyst-comprising-layer-side shielding portions can also be usedas a spacer to maintain the above space at a constant width. Moreover,when the height of such portion as a spacer is insufficient, anotherspacer may be separately provided at the shielding portions.

Specifically, when the above photocatalyst-comprising layer and celladhesiveness variation material layer are arranged so that the layersface each other with a predetermined space, the abovephotocatalyst-comprising-layer-side shielding portions and celladhesiveness variation material layer can be arranged in close contact.This makes it possible to precisely obtain the above predeterminedspace. Furthermore, through energy irradiation from thephotocatalyst-comprising-layer-side base plate under such state, itbecomes possible to precisely form a cell adhesiveness variation patternon the cell adhesiveness variation material layer.

A method for forming such photocatalyst-comprising-layer-side shieldingportions is not particularly limited. Such method is appropriatelyselected and used depending on the properties of the surface on whichphotocatalyst-comprising-layer-side shielding portions are formed,shielding property as required against energy, and the like.

For example, such photocatalyst-comprising-layer-side shielding portionsmay be formed by forming a metal thin film made of chrome or the likewith a thickness between approximately 1000 Å and 2000 Å by a sputteringmethod, a vacuum deposition method, or the like and then patterning thethin film. A general patterning method such as a sputtering method canbe used as such patterning method.

Furthermore, such patterning method may also be a method that comprisespreparing a layer that comprises shielding particles such as carbon fineparticles, metallic oxide, an inorganic pigment, or an organic pigmentin a resin binder and then patterning such layer. Examples of such resinbinder that is used herein include 1 type of or a mixture of 2 or moretypes of resins such as a polyimide resin, an acrylic resin, an epoxyresin, polyacryl amide, polyvinyl alcohol, gelatin, casein, andcellulose, and a photosensitive resin. Furthermore, an O/W emulsion typeresin composition such as an emulsified reactive silicone can be used.The thickness of each shielding portion made of resin can be determinedwithin a range between 0.5 μm and 10 μm. As a patterning method used forsuch shielding portions made of resin, a generally employed method suchas a photolithography method, printing method, or the like can be used.

Two possible positions of photocatalyst-comprising-layer-side shieldingportions are explained in the above explanation. One of such position isbetween a base body and a photocatalyst-comprising layer. The other oneis the surface of a photocatalyst-comprising layer. In addition to suchpositions, an embodiment that can also be employed comprises formingphotocatalyst-comprising-layer-side shielding portions on the surface ofa base body on the side where no photocatalyst-comprising layer isformed. In this embodiment, a photomask may be contacted closely butremovably to such surface, for example. Such embodiment can beappropriately used for a case where a cell adhesiveness variationpatterns is changed between small lots.

d. Primer Layer

Next, a primer layer that is used for aphotocatalyst-comprising-layer-side base plate in this embodiment willbe explained. In this embodiment, whenphotocatalyst-comprising-layer-side shielding portions are patterned ona base body as described above and then a photocatalyst-comprising layeris formed thereon, so as to prepare aphotocatalyst-comprising-layer-side base plate, a primer layer may beformed between the above photocatalyst-comprising-layer-side shieldingportions and photocatalyst-comprising layer.

The action and functions of such primer layer are not always clear.Through the formation of the primer layer betweenphotocatalyst-comprising-layer-side shielding portions and aphotocatalyst-comprising layer, it is thought that the primer layerexhibits a function to prevent the diffusion of impurities (that arefactors that inhibit variation in cell adhesiveness of a celladhesiveness variation material layer caused by the action of aphotocatalyst) coming from inside of thephotocatalyst-comprising-layer-side shielding portions or each openingexisting between the photocatalyst-comprising-layer-side shieldingportions. Particular examples of such impurities include residues andimpurities such as metal and metal ions that are generated at the timeof patterning of the photocatalyst-comprising-layer-side shieldingportions. Therefore, by the formation of such primer layer, treatmentfor causing variation in cell adhesiveness can proceed with highsensitivity. As a result, it becomes possible to obtain a pattern withhigh resolution.

In addition, such primer layer in this embodiment prevents impurities(existing not only on the photocatalyst-comprising-layer-side shieldingportions but also at an opening formed between suchphotocatalyst-comprising-layer-side shielding portions) from affectingthe action of a photocatalyst. The primer layer is preferably formed onthe whole surface of the photocatalyst-comprising-layer-side shieldingportions including the opening.

Such primer layer in this embodiment is not particularly limited, aslong as it is formed such that photocatalyst-comprising-layer-sideshielding portions of a photocatalyst-comprising-layer-side base plateand a photocatalyst-comprising layer do not contact with each other.

A material composing such primer layer is not particularly limited. Aninorganic material hardly decomposed by the action of a photocatalyst ispreferred. A specific example of such material is amorphous silica. Whensuch amorphous silica is used, a precursor of such amorphous silica is asilicon compound represented by general formula SiX₄, where X indicateshalogen, a methoxy group, an ethoxy group, an acetyl group, or the like.Hydrolysates of such compound, such as silanol or polysiloxane with anaverage molecular weight of 3000 or less, are preferable.

Furthermore, the film thickness of such primer layer is preferablywithin the range between 0.001 μm and 1 μm and particularly preferablywithin the range between 0.001 μm and 0.1 μm.

(2) Energy Irradiation

Next, energy irradiation in this step will be explained. In thisembodiment, the above cell adhesiveness variation material layer and theabove photocatalyst-comprising layer of the abovephotocatalyst-comprising-layer-side base plate are arranged so that thelayers face each other, and then energy irradiation is carried out froma predetermined direction. Thus, a pattern with variation in celladhesiveness of the cell adhesiveness variation material layer can beformed.

The above expression “are arranged” means a state where the layers arearranged so that a photocatalyst substantially acts on the surface ofthe cell adhesiveness variation material layer. The term also means, inaddition to a state where the layers are caused to come into actual andphysical contact, a state where the above photocatalyst-comprising layerand the above cell adhesiveness variation material layer are arrangedwith a predetermined space. Such space is preferably 200 μm or less.

The above space in this embodiment is particularly within the rangebetween 0.2 μm and 10 μm and preferably within the range between 1 μmand 5 μm in consideration of extremely good patterning accuracy, highphotocatalyst sensitivity, and good efficiency of causing variation inthe cell adhesiveness of a cell adhesiveness variation material layer.The space within such range is particularly effective for a celladhesiveness variation material layer with a small area that enablescontrol of such space with particularly high accuracy.

On the other hand, when a cell adhesiveness variation material layerwith an area that is as large as 300 mm×300 mm or greater, for example,is treated, it is extremely difficult to form a fine space as describedabove between a photocatalyst-comprising-layer-side base plate and acell adhesiveness variation material layer without causing them to comeinto contact. Therefore, when a cell adhesiveness variation materiallayer has relatively a large area, the above space is preferably withina range between 10 μm and 100 μm and particularly preferably within therange between 10 μm and 20 μm. The space set to be within such range canhave effects of: causing no problems such as lowered patterning accuracy(e.g., a blur patterning), deteriorated photocatalyst sensitivity, andlower efficiency of causing variation in cell adhesiveness as a resultof such deteriorated sensitivity; and not generating uneven variation inthe cell adhesiveness on a cell adhesiveness variation material layer.

When such cell adhesiveness variation material layer with a relativelylarge area is subjected to energy irradiation, within an apparatus forenergy irradiation, the space between aphotocatalyst-comprising-layer-side base plate and a cell adhesivenessvariation material layer is preferably set in an apparatus forpositioning the plate and the layer within the range between 10 μm and200 μm and particularly preferably within the range between 10 μm and 20μm. Determination of the space within such a range makes it possible toarrange a photocatalyst-comprising-layer-side base plate and a celladhesiveness variation material layer without causing any drasticdecrease in patterning accuracy or in photocatalyst sensitivity, andwithout the two coming into contact.

A photocatalyst-comprising layer and the surface of a cell adhesivenessvariation material layer are arranged with a predetermined space asdescribed above. Thus, removal of active oxygen species generated by theaction of oxygen, water, and a photocatalyst is facilitated.Specifically, when the space between a photocatalyst-comprising layerand a cell adhesiveness variation material layer is narrower than thosewithin the above ranges, it becomes difficult to remove the above activeoxygen species. As a result, the rate of causing variation in celladhesiveness may be lowered. Thus, such narrow space is not preferable.Furthermore, arrangement with a space wider than those within the aboveranges makes it difficult for the generated active oxygen species toreach the cell adhesiveness variation material layer. This case is alsonot preferable because this may result in a lower rate of causingvariation in cell adhesiveness.

An example of a method for arranging a photocatalyst-comprising layerand a cell adhesiveness variation material layer with uniform andextremely narrow space is a method that uses a spacer. A uniform spacecan be formed with the use of a spacer. Furthermore, portions (of thesurface of a cell adhesiveness variation material layer) to which thespacer is caused to come into contact are free from the action of aphotocatalyst. Hence, the spacer is prepared to have a pattern similarto that of the above cell adhesiveness variation pattern, so that apredetermined cell adhesiveness variation pattern can be formed on acell adhesiveness variation material layer.

In this embodiment, such arrangement should be maintained at leastduring energy irradiation.

Types of energy to be used for irradiation, irradiation method, theamount of energy irradiation, and the like are similar to thoseexplained in the above 1^(st) embodiment.

In addition, the present invention is not limited to the aboveembodiments. The above embodiments are provided for illustrativepurposes. Any embodiment that has substantially the same constitution asthat of the technical idea disclosed in the claims of the presentinvention and exerts action and effects similar to those exerted by thepresent invention is encompassed within the technical scope of thepresent invention.

III. Adhesion of Angiogenic Cells to Cell Array Substrate

In the cell array substrate, angiogenic cells are caused to adhere tothe regions having good cell adhesiveness of the above cell arraysubstrate having a cell adhesiveness variation pattern that comprisesregions having different cell adhesiveness. The cell array substrate ofthe present invention has such cell adhesiveness variation pattern thatcomprises regions having good cell adhesiveness and regions havinginhibited cell adhesiveness, as described above. Hence, when cells areuniformly inoculated on the surface of the cell array substrate and thensubjected to incubation for a predetermined time, the thus obtained cellarray substrate has a cell pattern formed to comprise regions havinggood cell adhesiveness, to which the cells adhere, and regions havinginhibited cell adhesiveness, to which no cells adhere. At such time, thesubstrate is subjected to liquid cleaning after incubation, so that thecells that weakly adhere to the substrate can be removed and a clearercell pattern can be obtained.

A culture sample containing angiogenic cells is preferably previouslysubjected to diffusion treatment by which a biological tissue is finelyfragmented and diffused in a liquid, or it is subjected to separationtreatment by which cells other than the target cells and othersubstances inhibiting the relevant experiment in a biological tissue areremoved.

Prior to inoculation of cells on the cell array substrate, it ispreferable to increase the number of the target cells throughpreliminary culture of the cells contained in a culture sample by anyone of a variety of culture methods. Examples of a general method thatcan be employed for such preliminary culture include a monolayer culturemethod, a coated dish culture method, and a gel culture method.Regarding preliminary culture, as a culture method that comprisescausing cells to adhere to the surface of a support and then culturingthe cells, the monolayer culture method is already known. Specifically,for example, when a culture sample and a culture solution are placed ina culture container and then maintained under certain environmentalconditions, specific viable cells alone will grow while adhering to thesurface of a support such as the culture container. The apparatuses,treatment conditions, and the like to be used herein are employedaccording to the general monolayer culture method and the like. As amaterial employed for the surface of a support to which cells adhere andgrow, a material with which cell adhesion and cell growth can besuccessfully carried out is selected. Furthermore, a chemical substance(namely, a cell adhesion factor) with which cell adhesion and cellgrowth can be successfully carried out is previously applied to thesurface of a support.

After culture, the culture solution within the culture container isremoved, thereby removing unnecessary components that do not adhere tothe surface of the support in the culture sample. Hence, only the viablecells adhering to the surface of the support can be harvested. Meanssuch as EGTA-trypsinization can be applied for harvesting viable cellsadhering to the surface of the support.

The above preliminarily cultured cells are inoculated on the cell arraysubstrate in a culture solution. A method for cell inoculation and aninoculation amount are not particularly limited. For example, a methoddisclosed in “Tissue Culture Technology (Soshiki Baiyo no Gijutu)”(edited by The Japanese Tissue Culture Association, pp. 266 to 270,issued by Asakura Pub. Co., 1999) can be used. It is preferable toinoculate cells in a sufficient amount so that the cells are notrequired to grow on the cell array substrate and so that the cellsadhere to the substrate in the form of monolayer. This is because tissueformation by cells is inhibited when cells aggregate, and even whencells are transferred to and then cultured on a basal membrane layer,their functions will be impaired. Specifically, approximately 2×10⁵cells are inoculated per 400 mm².

It is preferable to cause cells to adhere to regions having good celladhesiveness through incubation of the cells that have been inoculatedon a cell array substrate in a culture solution. As a culture solution,a medium that is generally used in the technical field can be used.According to the cell types to be used herein, a basic medium disclosedin “Tissue Culture Technology (Soshiki Baiyo no Gijutu)” (edited by TheJapanese Tissue Culture Association, issued by Asakura Pub. Co., 3rded., p. 581) can be used. Examples of such medium include an MEM medium,a BME medium, a DME medium, an αMEM medium, an IMEM medium, an ESmedium, a DM-160 medium, Fisher medium, an F12 medium, a WE medium, andan RPMI medium. Furthermore, one of these media supplemented with aserum component (e.g., fetal calf serum) or the like, a commercial serumfree medium, and the like can be used.

Time for incubation is generally between 30 minutes and 48 hours andpreferably between 4 hours and 24 hours. When cells are incubated for aproper time period and then the substrate is washed, cells adhere toregions of the cell array substrate having good cell adhesiveness but donot adhere to regions of the cell array substrate having inhibited celladhesiveness. Also, it becomes possible to easily transfer the remainingcells to a basal membrane layer.

The temperature for incubation differs depending on the types of cellsto be caused to adhere, and it is generally 37° C. Cells are preferablyincubated under a CO₂ atmosphere using a CO₂ cell culture incubator.After incubation, the cell array substrate is washed, so as to wash offcells that have not adhered to the substrate. Thus, the cells can bearrayed in a pattern.

FIG. 7 shows an embodiment of the step of causing angiogenic cells toadhere to a cell array substrate in a pattern and then transferring theadhered cells to a basal membrane layer. Angiogenic cells are inoculatedon a cell array substrate (15) comprising regions having good celladhesiveness (17) and regions having inhibited cell adhesiveness (18)patterned thereon. The cells are then caused to adhere to form apattern. Subsequently, the cell array substrate to which the angiogeniccells adhere is caused to come into close contact with a basal membranelayer that is provided covering almost the entire surface of a region ofa tissue-forming cell layer on which a blood vessel network will beformed, so as to transfer and culture the cells. If necessary, the cellsare stimulated with a cell stimulating factor (22). In addition, asshown in the figure, the basal membrane layer is not necessarily formedcovering the entire surface of the tissue-forming cell layer, and it maybe formed covering a region to which a blood vessel network will betransferred.

BEST MODE OF CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail byreferring to examples, but the present invention is not limited by theseexamples.

Example 1 Preparation a Cell Array Substrate

1.5 g of fluoroalkyl silane TSL8233 (GE Toshiba Silicones), 5.0 g oftetramethoxysilane TSL8114 (GE Toshiba Silicones), and 2.4 g of5.0×10⁻³N HCl were mixed for 12 hours and then diluted 10-fold withisopropyl alcohol.

Next, 2.0 g of the solution was applied to a 10 cm×10 cm soda glasssubstrate using a spin coater at 1000 rpm for 5 seconds. The substratewas dried at 150° C. for 10 minutes.

Next, 3.0 g a titanium oxide sol solution (ISHIHARA SANGYO KAISHA, LTD.STK-03) diluted 3-fold with isopropyl alcohol was used as a compositionfor a photocatalyst-comprising layer.

The above composition for a photocatalyst-comprising layer was appliedto the patterned surface (on which line portions each having a width of60 μm and space portions each having a width of 400 μm had been arrangedalternately) of a line & space negative photomask (quartz) using a spincoater at 700 rpm for 3 seconds, followed by 10 minutes of dryingtreatment at 150° C. Thus, a photomask comprising a transparentphotocatalyst-comprising layer was formed.

The above photocatalyst-comprising layer surface of the photomask andthe above cell adhesiveness variation material layer surface of thesubstrate were arranged with a space of 10 μm between the surfaces. UVexposure was carried out from the photomask side using a mercury lamp(wavelength: 365 nm) with an illuminance of 25.0 mW/cm² for apredetermined time. The thus obtained cell array substrate had a celladhesiveness variation pattern wherein linear regions having good celladhesiveness and each having a width of 60 μm and the spaces comprisingregions having inhibited cell adhesiveness and each having a width of400 μm had been arranged alternately.

Example 2 Culture and Adhesion of Vascular Endothelial Cells onto theCell Array Substrate

As cells to be cultured, bovine carotid-derived vascular endothelialcells (Onodera M, Morita I, Mano Y, Murota S: Differential Effects ofNitric Oxide on the Activity of Prostaglandin Endoperoxide h Synthase-1and -2 in Vascular Endothelial Cells, Prostag Leukotress 62: 161-167,2000) of 5th to 20th generations obtained by successive culture wereused.

Bovine carotid-derived vascular endothelial cells that had reachedconfluence in a 10 cm dish were removed by 0.05% trypsin-EDTA treatment.The number of cells was counted using a Coulter Counter™ ZM and then theconcentration was adjusted to 10⁶ cells/ml. The cell array substrate(exposure time: 360 seconds) prepared in Example 1 was sterilized withan autoclave. The cell array substrate was placed on the culture dish(Heraeus Quadriprem™, 76 mm×26 mm, and 1976 mm²) containing a culturesolution (MEM medium comprising 5% fetal calf serum) and then the aboveendothelial cells were inoculated at 10⁶ cells/5 ml per well. The cellswere incubated for 24 hours using a CO² incubator.

A carbocyanine fluorescent dye (DiI, Invitrogen Corp.) was dissolved inan MEM medium comprising 5% fetal calf serum at a concentration of 10μg/ml. The above cell array substrate on which the cells had beenarrayed was immersed in the medium, followed by 1 hour of culture at 37°C. Subsequently, the cell array substrate was returned in an MEM mediumcomprising 5% fetal calf serum.

Example 3

i) Mouse hepatic parenchymal cells were harvested and then cultured on acommercial 96-well NIPAAm (poly-N-isopropylacrylamide) plate. Afterstaining with a carbocyanine fluorescent dye (DiO, Invitrogen Corp.)with the above concentration, the medium containing the dye was replacedwith an MEM medium comprising 5% fetal calf serum.

GFR Matrigel (Becton, Dickinson and Company) was diluted 10-fold with anMEM medium comprising 5% fetal calf serum. The medium containing the gelwas added at 25 μl per well of the plate where mouse hepatic parenchymalcells had been cultured, followed by 1 hour of culture at 37° C. Thus, abasal membrane layer comprising a gel thin film layer was formed on thecells.

ii) The substrate prepared in Example 2, on which vascular endothelialcells had been arrayed, was immersed in thehepatic-parenchymal-cell-cultured plate prepared in i), so as to causethe basal membrane layer on the hepatic parenchymal cells to come intocontact with vascular endothelial cells. The cells were cultured at 37°C. for 24 hours and then the cell array substrate was removed(separated).iii) The plate was shaken at approximately 20° C. for 30 minutes, lightpipetting was carried out, and then the thus formed tissue construct wasremoved using forceps.iv) An immunodeficient mouse was anesthetized, the dorsal region wasincised, and the tissue construct prepared through i) to iii) wastransplanted into the mouse liver. The transplanted site was sutured. Onday 1 and day 3 after suturing, the transplanted site was incised again,and then the transplanted tissue was observed under a confocal lasermicroscope (DiI→excitation wavelength of 530 nm/observance wavelength of590 nm and DiO→excitation wavelength of 480 nm/observance wavelength of510 nm).Results

The growth of the transplanted hepatic parenchymal cells was confirmedby observation at an excitation wavelength of 480 nm. Furthermore,transplanted vascular endothelial cells were observed at an excitationwavelength of 530 nm. Thus, it was confirmed that capillary vessels hadbeen formed in the same pattern as the previously formed pattern ofvascular endothelial cells.

Example 4

Vascular endothelial cells were arrayed on a cell array substrateaccording to procedures similar to those used in Examples 1 and 2.Furthermore, the steps i) to iii) of Example 3 were carried out, andtissue constructs each comprising hepatic parenchymal cells, a basalmembrane, and blood vessels were prepared.

Next, 3, 5, and 7 pieces of the tissue constructs thus prepared werelaminated together, so as to prepare laminated tissue constructs.Immediately after lamination, the tissue constructs were eachtransplanted into the livers of immunodeficient mice. Each transplantedsite was sutured. On day 1 and day 3 after suturing, each transplantedsite was incised again and then the transplanted tissue was observed.

Results

The growth of the transplanted hepatic parenchymal cells was confirmedby observation at an excitation wavelength of 480 nm. Furthermore,transplanted vascular endothelial cells were observed at an excitationwavelength of 530 nm. Thus, it was confirmed that capillary vessels hadbeen formed in the same pattern as the previously formed pattern of thevascular endothelial cells. The amounts of hemoglobin in the excisedtissues were analyzed. As a result, it was confirmed that the amount ofhemoglobin in each transplanted site was equivalent to that innon-transplanted sites of the mouse liver.

Comparative Example 4

Tissue constructs each comprising hepatic parenchymal cells and a basalmembrane layer were formed according to the steps i) to iii) of Example3. Next, 3, 5, and 7 pieces of the tissue constructs thus prepared werelaminated together, so as to form laminated tissue constructs.Immediately after lamination, the laminated tissue constructs were eachtransplanted into the livers of immunodeficient mice. The transplantedsites were sutured. On day 1 and day 3 after suturing, each transplantedsite was incised again and then the transplanted tissue was observed.

Results

In the cases where 5 or 7 pieces of tissue constructs had been laminatedtogether, the necrosis of the transplanted hepatic parenchymal cells wasobserved at an excitation wavelength of 480 nm. Furthermore, the amountsof hemoglobin in the excised tissues were analyzed. As a result, almostno hemoglobin was observed at the transplanted sites.

Example 5

Vascular endothelial cells were arrayed on a cell array substrateaccording to procedures similar to those used in Examples 1 and 2.Furthermore, the steps i) to iii) of Example 3 were carried out, so asto form 1^(st) tissue constructs each comprising hepatic parenchymalcells, a basal membrane, and blood vessels. Next, 2^(nd) tissueconstructs each comprising hepatic parenchymal cells and a basalmembrane layer were formed according to the steps i) and iii) of Example3.

3 pieces of the thus formed 1^(st) tissue constructs and 3 pieces of thethus formed 2^(nd) tissue constructs (total 6 tissue constructs) werelaminated together alternately, so as to form laminated tissueconstructs. Immediately after lamination, the laminated tissueconstructs were each transplanted to the livers of immunodeficient mice,and then the transplanted sites were sutured. On day 1 and day 3 aftersuturing, each transplanted site was incised again, and then thetransplanted tissue was observed.

The growth of the transplanted hepatic parenchymal cells was confirmedby observation at an excitation wavelength of 480 nm. Furthermore,transplanted vascular endothelial cells were observed at an excitationwavelength of 530 nm. Thus, it was confirmed that capillary vessels hadbeen formed in the same pattern as the previously formed pattern of thevascular endothelial cells. The amounts of hemoglobin in the excisedtissues were analyzed. As a result, it was confirmed that the amount ofhemoglobin in each transplanted site was half or more of the amountresulting in Example 4.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, an artificial tissue construct thathas means for transporting nutrients, oxygen, waste products, or thelike and is viable in vivo can be provided.

The invention claimed is:
 1. A method for producing a tissue constructcomprising a tissue-forming cell layer, a basal membrane layer, and avascular layer, which comprises the steps of: (a) forming atissue-forming cell layer on a culture base; (b) forming a basalmembrane layer on the obtained tissue-forming cell layer; (c) forming avascular layer, which comprises the steps of: (i) providing a cell arraysubstrate having a cell adhesiveness variation pattern that comprisesregions having good cell adhesiveness and regions having inhibited celladhesiveness, (ii) providing angiogenic cells to the cell arraysubstrate, wherein the angiogenic cells adhere to the regions havinggood cell adhesiveness on the substrate, thereby forming a vascularlayer having a patterned form; (iii) recovering the vascular layer fromsaid cell array substrate in a manner such that the vascular layerretains its patterned form; (d) transferring the vascular layer in itspatterned form onto the basal membrane layer; (e) culturing thetransferred cells; and (f) separating and collecting a tissue constructcomprising the tissue-forming cell layer, the basal membrane layer, andthe vascular layer from the culture base.
 2. The method according toclaim 1, wherein the cell adhesiveness variation pattern is formed byarranging the cell adhesiveness variation layer that comprises the celladhesiveness variation material and the photocatalyst-comprising layerso that the layers face each other, and then carrying out energyirradiation.
 3. The method according to claim 1, wherein the celladhesiveness variation pattern is a pattern wherein linear regionshaving good cell adhesiveness and spaces comprised of the regions havinginhibited cell adhesiveness are arranged alternately, the line widths ofthe regions having good cell adhesiveness are each between 20 μm and 200μm, and the space widths between such lines are each between 100 μm and1000 μm.
 4. The method according to claim 1, wherein the culture basehas a surface that is capable of retaining cells with weak adhesiveness.5. A method for producing a laminated tissue construct, which compriseslaminating together tissue constructs produced by the method accordingto claim
 1. 6. The method according to claim 5, which further comprisesthe step of transporting a culture solution to the vascular layer withinthe laminated tissue construct.
 7. The method according to claim 5,wherein the cell adhesiveness variation pattern is formed by arranging acell adhesiveness variation layer that comprises a cell adhesivenessvariation material and a photocatalyst-comprising layer so that thelayers face each other, and then carrying out energy irradiation.
 8. Themethod according to claim 5, wherein the cell adhesiveness variationpattern is a pattern wherein linear regions having good celladhesiveness and spaces comprised of the regions having inhibited celladhesiveness are arranged alternately, the line widths of the regionshaving good cell adhesiveness are each between 20 μm and 200 μm, and thespace widths between such lines are each between 100 μm and 1000 μm. 9.The method according to claim 5, wherein the culture base has a surfacethat is capable of retaining cells with weak adhesiveness.
 10. A methodfor producing a laminated tissue construct which comprises producing afirst tissue construct by a method comprising the following steps(a)-(f): (a) forming a tissue-forming cell layer on a culture base; (b)forming a basal membrane layer on the obtained tissue-forming celllayer; (c) forming a vascular layer, which comprises the steps of: (i)providing a cell array substrate having a cell adhesiveness variationpattern that comprises regions having good cell adhesiveness and regionshaving inhibited cell adhesiveness, (ii) providing angiogenic cells tothe cell array substrate, wherein the angiogenic cells adhere to theregions having good cell adhesiveness on the substrate, thereby forminga vascular layer having a patterned form; (iii) recovering the vascularlayer from said cell array substrate in a manner such that the vascularlayer retains its patterned form; (d) transferring the vascular layer inits patterned form onto the basal membrane layer; (e) culturing thetransferred cells; and (f) separating and collecting a tissue constructcomprising the tissue-forming cell layer, the basal membrane layer, andthe vascular layer from the culture base, producing a second tissueconstruct by a method comprising the following steps (g)-(i): (g)forming a tissue-forming cell layer on a culture base; (h) forming abasal membrane layer on the obtained tissue-forming cell layer; and (i)separating and collecting the tissue-forming cell layer and the basalmembrane layer from the culture base, and laminating together the firstand the second tissue constructs.
 11. The method according to claim 10,which further comprises the step of transporting a culture solution tothe vascular layer within the laminated tissue construct.
 12. The methodaccording to claim 10, wherein the cell adhesiveness variation patternis formed by arranging a cell adhesiveness variation layer thatcomprises a cell adhesiveness variation material and aphotocatalyst-comprising layer so that the layers face each other, andthen carrying out energy irradiation.
 13. The method according to claim10, wherein the cell adhesiveness variation pattern is a pattern whereinlinear regions having good cell adhesiveness and spaces comprised of theregions having inhibited cell adhesiveness are arranged alternately, theline widths of the regions having good cell adhesiveness are eachbetween 20 μm and 200 μm, and the space widths between such lines areeach between 100 μm and 1000 μm.
 14. The method according to claim 10,wherein the culture base has a surface that is capable of retainingcells with weak adhesiveness.